Vaccine

ABSTRACT

The invention relates to a vaccine for the treatment of disease caused by  Neisseria , the vaccine comprising o   more immunogenic components for  Neisseria  serogroups, as well as antibodies to the immunogenic components and m   preventing and treating  Neisseria  infections. The immunogens are based on elements of the inner core lipopolysacch

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application entitled“Vaccine”, filed Jul. 24, 2009 and assigned Ser. No. 12/508,683, whichis a divisional of U.S. patent application entitled “Vaccine”, filed onJul. 11, 2002 and assigned Ser. No. 10/089,583, now U.S. Pat. No.7,585,510, which claimed the benefit of PCT/GB00/03758 filed Oct. 12,2000, which claimed the benefit of U.S. provisional patent application60/196,305, filed Apr. 12, 2002 and U.S. provisional patent application60/156,940, filed Sep. 30, 1999.

TECHNICAL FIELD

The present invention relates to vaccines against Neisseria infection,especially to infection by pathogenic Neisseria meningitidis andNeisseria gonorrhoeae.

BACKGROUND OF THE INVENTION

Septicaemia and meningitis caused by Neisseria meningitidis remain aglobal health problem, especially in young children. Neisseriameningitidis is usually a commensal of the nasopharynx, the only majornatural reservoir of this organism. The virulence factors thatpotentiate the capacity of Neisseria meningitidis to cause invasivedisease include capsular polysaccharides, pili (fimbrae) or outermembrane proteins and lipopolysaccharides (DeVoe, I. W. 1982. Microbiol.Rev. 46: 162-190; Jennings, H. J. 1989. Contrib. Microbiol. Immunol. 10:151-165; Tonjum, T., and M. Koomey. 1997. Gene 192: 155-163; Nassif, X.,et al., 1997. Gene 192: 149-153; Poolman, J. T. 1996. Adv. Exp. Med.Biol. 397: 73-33; Verheul, A. F., et al., 1993. Microbiol. Rev. 57:34-49; Preston, A, et al., 1996 Crit. Rev. Microbiol. 22: 139-180).

Existing licensed vaccines against capsular serogroups A, C, W and X areavailable (Frasch, C. E. 1989. Clin. Microbiol. Rev. 2 Suppl: S134-138;Herbert, M. A., et al., 1995. Commun. Dis. Reg. CDR Rev. 5: R130-135;Rosenstein, N., et al., 1998. J.A.M.A. 279: 435-439), but generally lacksatisfactory immunogenicity in very young children and do not inducelong lasting protective immunity (Peltola, H., et al., 1977. New Engl.J. Med. 297: 686-691; Peltola, H., et al., 1985. Pediatrics 76: 91-96;Reingold, A. L., et al., 1985. Lancet 11:114-118; Lepow, M. L., et al.,1986. J. Infect. Dis. 154: 1033-1036; Cadoz, M. 1998. Vaccine 16:1391-1395). Nonetheless, their utility has been significant in affordingprotection to selected populations such as the military, travelers andthose at exceptional risk in outbreaks or epidemics (CDC. 1990. MMWRMorb. Mortal. Wkly. Rep. 39, No. 42: 763). Very recently, meningococcalconjugate Group C vaccines have been introduced as a routineimmunization in the United Kingdom.

The major public health priority concerning invasive meningococcalinfections is to identify Group B vaccines that are highly effective ininfants and give long term protection. Group B strains have accountedfor a substantial, often a majority of invasive Neisseria meningitidisinfections in many countries in Europe and North America (CDR. 1997April. Communicable Disease Weekly Report. 7, No. 14). Prevention ofGroup B invasive disease represents a particularly difficult challengein vaccine development as the capsular polysaccharide is very poorlyimmunogenic and even conjugates have shown disappointing immunogenicity(Jennings, H. J., and H. C. Lugowski. 1981. J. Immunology 127:1011-1018). Further, there are concerns about the safety of vaccineswhose rationale is to induce antibodies to the Group B polysaccharide, ahomopolymer of α-linked 2-8 neuraminic acid. The identicalpolysialicacid (PSA) is a post translational modification of aglycoprotein present on human cells, especially neurons, the latter isreferred to as neural cell adhesion molecule (N-CAM) (Finne, J., et al.,1983. Lancet 2: 355-357). Both theoretical and experimental evidencehave been used to argue that the induction of antibodies might result inautoimmune, pathological damage to host tissues.

Alternative approaches to develop vaccine candidates against Group BNeisseria meningitidis are being actively explored. These include: outermembrane porins (Poolman, J. T., et al., 1995. Meningococcal disease, p.21-34K. Cartwright (ed.). John Wiley and sons, Wetzler, L. M. 1994. Ann.N.Y. Acad. Sci. 730: 367-370; Rosenquist E., et al., 1995. Infect.Immun. 63:4642-4652; Zollinger, W. D., et al., 1997. Infect. Immun. 65:1053-1060), transferrin binding proteins (Al'Aldeen, A. A., and K. A.Cartwright. 1996. J. Infect. 33: 153-157) and lipopolysaccharides(Verheul, A. F., et al., 1993. Infect. Immun. 61: 187-196; Jennings, H.J., et al., 1984. Infect. Immun. 43: 407-412; Jennings, H. J., et al.,987. Antonie Van Leeuwenhoek 53: 519-522; Gu, X. X., and C. M. Tsai.1993. Infect. Immun. 61: 1873-1880; Moxon, E. R., et al., 1998. Adv.Exp. Med. Biol. 435: 237-243).

The structure of Neisseria meningitidis LPS has been studied inconsiderable detail by Jennings H. and co-workers with additionalcontributions by others (Griffiss, J. M. et al., 1987 Infect. Immun. 55:1792-1800; Stephens, D. S., et al., 1994. Infect. Immun. 62: 2947-2952;Apicella, M. A., et al., 1994. Methods Enzymol. 235: 242-252; Poolman,J. T. 1990. Polysaccharides and membrane vaccines, p. 57-86. inBacterial Vaccines, A. Mizrahi (ed.)., et al. 1997. FEMS Microbiol Lett.146: 247-253). The structures of major glycoforms for severalimmunotypes (L1-L9) have been published L1, L6 (Di Fabio, J. L., et al.,1990. Can. J. Chem. 68: 1029-1034; Wakarchuk, W. W., et al., 1998. Eur.J. Biochem. 254: 626-633); L3 (Pavliak, V., et al., 1993. J. Biol. Chem.268: 14146-14152); L5 (Michon, F., et al. 1990. J. Biol. Chem.265:7243-7247); L2 (Gamian, A., et al., 1992. J. Biol. Chem. 267:922-925); L4, L7 (Kogan, G., et al., 1997. Carbohydr. Res. 298:191-199): L8 (Wakarchuk, W. W., et al., 1996, J. Biol. Chem. 271,19166-19173), L9 (Jennings, H. J., et al., 1983. Carbohydr. Res. 21:233-241). Reference is also made to the following discussion of theaccompanying FIG. 1.

It is known that, in addition to this inter-strain variation, individualNeisseria meningitidis strains exhibit extensive phase variation ofouter core LPS structures (reviewed in van Putten, J. P., and B. D.Robertson. 1995. Mol. Microbiol. 16: 847-853 and Andersen, S. R., etal., 1997. Microb. Pathog. 23: 139-155). The molecular mechanism of thisintra strain variation involves hypermutable loci within the readingframes encoding several glycosyl transferases (Gotschlich, E. G. 1994.J. Expt. Med. 180: 2181-2190, Jennings, M. P., et al., 1995. Mol.Microbiol. 18: 729-740). Similar mechanisms of phenotypic variation havebeen reported for other phase-variable surface components of pathogenicNeisseria, including Opc (Sakari, J., et al., 1994. Mol. Microbiol. 13:207-217), Opa (Stem, A., et al., 1986. Cell 47: 61-71) and PiIC proteins(Jonsson, A. B., et al., 1991. EMBO. J. 10: 477-488). The highfrequency, reversible molecular switching is mediated by homopolymerictracts of cytosines or guanines through slippage-like mechanisms thatresults in frame shifts (Gotschlich, E. C. 1994. J. Expt. Med. 180:2181-2190, Jennings, M. P., et al., 1995. Mol. Microbiol. 18: 729-740;Stern, A. and T. F. Meyer. 1987. Mol. Microbiol. 1: 5-12).

Despite the extensive antigenic variation of LPS, the inner core of theLPS has been considered to be relatively highly conserved, and thereforethe use of the inner core of the LPS structure has been suggested foruse in vaccine design. However, the problems with candidate vaccinegeneration in this way are numerous.

First, although it was known that certain components of the inner corecould be immunogenic (Jennings, H. J. et al., 1984. Infect. Immun. 43:407-412; Verheul, A. F., et al., 1991. Infect. Immun. 59: 3566-3573),the extent of conservation of these epitopes across the diversity ofmeningococcal disease isolates was not known and evidence ofbactericidal activity of antibodies to these epitopes has not beenshown. U.S. Pat. No. 5,705,161 discloses that oligosaccharides ofmeningococcal immunotypes differ, for example, with regard tomonosaccharide composition, amount and location of phosphoethanolaminegroups and degree of acetylation of the inner core GlcNAc unit or otherunits, indicating that many possible structures may be found in the corestructure. U.S. Pat. No. 5,705,161 also suggests that a portion of thecore of a meningococcal LPS may be suitable for use in a vaccine,although no specific immunogenic epitopes or supporting data aredisclosed.

Secondly, given the presence of the outer core LPS structure and othersurface exposed non-LPS structures, including capsule, it is not knownwhether the inner core structure is accessible to the immune system toallow a bactericidal immune response to be generated. Furthermore, anyvaccine would need to contain immunogenic structures which elicit animmune response to the complete range of pathogenic Neisseriameningitidis strains. However, the extent of variation exhibited by theinner core structure of virulent strains is not known, and rigorousinvestigation of the problem has not been undertaken.

Furthermore, in the publication New Generation Vaccines (1997, Ed. M. M.Levine, publ. Marcel Deker Inc, New York, Chapter 34, page 481), it isstated that, with respect to vaccine development, “including LPS thatconsists only of the common inner core region of the oligosaccharide maynot result in induction of bactericidal antibodies.”.

In addition, other species of the genus Neisseria pose global healthproblems. For example, Neisseria gonorrhoeae is involved in sexuallytransmitted diseases such as urethritis, salpingitis, cervicitis,proctitis and pharyngitis, and is a major cause of pelvic inflammatorydisease in women.

Accordingly, there is still a need in the art for an effective vaccineagainst pathogenic Neisseria infection, such as Neisseria meningitidisand Neisseria gonorrhoeae infection.

The present invention sets out to address this need.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a vaccine for the treatmentof disease caused by Neisseria infection, the vaccine comprising animmunogenic component of Neisseria strains. The vaccine presents aconserved and accessible epitope that in turn promotes a functional andprotective response.

We have now discovered that the inner core of the LPS of Neisseria canbe used to generate a protective immune response to Neisseriainfections, for example Neisseria meningitidis infections. Forsimplicity the present invention is herein exemplified principally bydiscussion of vaccines and treatments against Neisseria meningitidisinfections, but the invention extends to diseases caused by otherpathogenic Neisseria species.

Using a range of novel monoclonal antibodies, epitopes belonging to theinner core of Neisseria meningitidis have been identified which havebeen found to be accessible to the immune system, and which are capableof stimulating the production of functional, protective antibodies.Moreover, analysis of Neisseria meningitidis strains using the newantibody tools indicates that certain epitopes are common to a range ofNeisseria meningitidis disease isolates, and sometimes occur in amajority of such strains. Accordingly a vaccine comprising only alimited range of Neisseria meningitidis inner core epitopes can provideeffective immunoprophylaxis against the complete range of strainscausing Neisseria meningitidis infection. Similar considerations applyto other pathogenic species.

In a related aspect, the invention provides a vaccine effective againststrains of the bacteria of the genus Neisseria, especially strains ofthe species Neisseria meningitidis. Particularly in the latter instance,the vaccine comprises one or more immunogens which can generateantibodies that recognize epitopes in encapsulated strains. The one ormore immunogens represent one or more accessible inner core epitopes.Thus, the immunogens can give rise to antibodies that recognize amajority of strains.

We use the word “principal” to refer to a majority. Thus, a principalimmunogenic component elicits antibodies to a majority of strains.

In our approach, antibodies were generated by immunizing mice usingNeisseria meningitidis galE mutants. The antibodies produced werespecific to the LPS inner core because galE mutants lack outer corestructures. The reactivity of these antibodies against a panel ofNeisseria meningitidis strains representative of the diversity found innatural populations of disease isolates was investigated. One monoclonalantibody reacted with 70% of all Neisseria meningitidis strains tested,suggesting strong conservation of the inner core epitope recognized bythis antibody, termed antibody B5. The epitope against which B5 reactshas been characterized and can be used to form the basis of a vaccine toprevent Neisseria infections.

A hybridoma producing the monoclonal antibody B5, designated hybridomaNmL3B5, has been deposited under the Budapest Treaty on 26 Sep. 2000with the International Depositary Authority of Canada in Winnipeg,Canada, and given the Accession Number IDAC 260900-1.

In this way, we have obtained proof in principle that one or more of theinner core epitopes of LPS are conserved and accessible to antibodies,that a specific immune response to these epitopes can mediateprotection, and that LPS inner core oligosaccharides can be candidatevaccines. The inner core LPS typically consists of an inner coreoligosaccharide attached to lipid A, with the general formula as shown:

where R1 is a substituent at the 3-position of HepII, and is hydrogen orGlc-α-(1, or phosphoethanolamine; R2 is a substituent at the 6-positionof HepII, and is hydrogen or phosphoethanolamine; R3 is a substituent atthe 7-position of HepII, and is hydrogen or phosphoethanolamine, and R4is acetyl or hydrogen at the 3-position, 4-position or 6-position of theGlcNAc residue, or any combination thereof; and where Glc isD-glucopyranose; Kdo is 3-deoxy-D-manno-2-octulosonic acid; Hep isL-glycero-D-manno-heptose, and GlcNAc is2-acetamido-2-deoxy-D-glucopyranose.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is now illustrated by the following Figures andExamples which are not limiting upon the present invention, wherein:

FIGS. 1A and 1B show representations of the structure of meningococcalLPS oligosaccharides of immunotypes L1-L9. Immunotypes are indicated tothe extreme left. The vertical dotted line marks the junction betweenthe inner core structures to the right and outer core structures to theleft. The epitope recognized by MAb B5 is indicated in bold (MAb B5positive). Arabic numerals indicate the linkage between sugars oramino-sugars. Alpha and beta indicate the carbon 1 linkage at thenon-reducing end of the sugar. Genes for incorporating each of the keysugars or amino-sugars into the LPS oligosaccharide in the biosyntheticpathway are indicated with arrows indicating where in the pathway thegene product is required. Abbreviations include: Kdo,2-keto-2-deoxyoctulosonic acid; PEtn, phosphoethanolamine; Gal,galactose: GLcNAc, N-acetyl glucosamine; Glc, glucose; Hep, Heptose.Immunotype L5 has no PEtn on the second heptose. The gene that adds theglucose to the second heptose (IgtG) is phase variable.

FIG. 1A illustrates the LPS structure of Neisseria meningitidisimmunotypes that are Mab B5 positive.

FIG. 1B illustrates the LPS structure of Neisseria meningitidisimmunotypes that are Mab B5 negative.

FIG. 2 illustrates cross reactivity of monoclonal antibody B5 withselected immunotypes and mutants of Neisseria meningitidis LPS.Cross-reactivity of MAb B5 with selected immunotypes and mutants ofNeisseria meningitidis LPS and O-deacylated (odA) LPS was determined bysolid phase ELISA. LPS glycoforms of immunotypes L2 (35E), L3 (H44/76),L4 (891), L5 (M981), L8 (M978), wild-type and respective mutants (galE,IgtA or IgtB), in a native or O-deacylated form, were coated onto ELISAplates (see methods) and reactivity of MAb B5 determined by standardELISA (OD A₄₅₀).

FIGS. 3A-3C illustrate space-filling 3-D molecular models of thecalculated (MMC) lowest energy states of the core oligosaccharide fromgalE mutants of L3, L4, and L8 dephosphorylated. Kdo moiety indicated ingrey is substituted at the 0-5 position by the heptose disaccharideinner core unit (red), HepI provides the point via a glucose residue(dark green) for extension to give α-chain epitopes, while HepII issubstituted by N-acetyl glucosamine residue (lighter green) at 0-2. PEtn(brown) is shown in 0-3 position in L3 immunotype and 0-6 in L4immunotype. Colour versions of this and the other figures for Example 1are to be found in Plested et al., 1999 Infect. Immunity 67, 5417-5426.

FIG. 3A illustrates the molecular model of the calculated (MMC) lowestenergy state of the core oligosaccharide from galE mutant of L3. Kdo ingrey, Heptose (Hep) in red, Glucose (Glc) and Glucosamine (GlcNAc) inlight and darker green, (PEtn) in brown.

FIG. 3B illustrates the molecular model of the calculated (MMC) lowestenergy state of the core oligosaccharide from galE mutant of L4. Kdo ingrey, Heptose (Hep) in red, Glucose (Glc) and Glucosamine (GlcNAc) inlight and darker green, (PEtn) in brown.

FIG. 3C illustrates the molecular model of the calculated (MMC) lowestenergy state of the core oligosaccharide from galE mutant of L8dephosphorylated. Kdo in grey, Heptose (Hep) in red, Glucose (Glc) andGlucosamine (GlcNAc) in light and darker green, (PEtn) in brown.

FIG. 4 illustrates cross reactivity of B5 with genetically modified L3LPS and chemically modified L8 LPS from Neisseria meningitidis asdetermined by solid phase ELISA. LPS glycoforms of immunotype L8 (M978)chemically modified by O-deacylation and HF treatment and immunotype L3H44/76) galE, icsB, icsA, lis; PB4 mutants (O-deacylated) were coatedonto ELISA plates (see methods) and reactivity of MAb B5 determined bystandard ELISA (OD_(A410nm)).

FIGS. 5A-5D illustrate confocal immunofluorescence microscopy ofNeisseria meningitidis organisms strain MC58 adherent to HUVECs.Confocal immunofluorescence microscopy of Neisseria meningitidisorganisms, strain MC58 adherent to human umbilical vein endothelialcells (HUVECs). FIG. 5A: Fluorescein tagging with rabbit polyclonalantibody specific for Group B Neisseria meningitidis capsule. FIG. 5B:rhodamine tagging of MAb B5, specific for galE LPS (×2400magnification). Confocal immunofluorescence microscopy of in vivo grownMC58 organisms stained as described in Plested et al., 1999. Infect.Immunity 67, 5417-5426. FIG. 5C: anti-capsular antibody. FIG. 5D: MAb B5(×2400 magnification).

FIGS. 6A and 6B illustrate silver stained tricine gels of LPSpreparations from Neisseria meningitidis group B strains not reactivewith MAb B5. These LPS preparations were either not treated (−) ortreated with (+) neuraminidase to show the presence of sialic acid:

FIG. 6A: Silver-stained tricine gel of LPS preparations (10 μg/lane)from Neisseria meningitidis Group B strains which were not reactive withMAb B5. These LPS preparations were either not treated (−) or treatedwith (+) neuraminidase to show the presence of sialic acid. MAb B5negative strains: Lanes 1, 2=NGE30; lanes 3, 4=BZ157; lanes 5, 6=EG328;lanes 7, 8=1000; lanes 9, 10=3906.

FIG. 6B: Silver-stained tricine gel of LPS preparations (10 μg/lane)from Neisseria meningitidis Group B strains which were not reactive withMAb B5. These LPS preparations were either not treated (−) or treatedwith (+) neuraminidase to show the presence of sialic acid. MAb B5negative strains: Lanes 1, 2=EG327; lanes 3.4=NGH38; lanes 5, 6=NGH15;MAb B5 positive strain: lanes 7, 8=MC58. Presence of sialic acid (NeuAc)indicated by +. This band was seen in untreated (−) and removed intreated (+) neuraminidase preparations.

FIGS. 7A-7C illustrates accessibility of the LPS epitope to A4 inNeisseria meningitidis whole cells. MAb A4 accesses the inner core LPSepitope in Neisseria meningitidis L4 galE mutant in the presence ofcapsule (magnification ×100). Neisseria meningitidis L4 galE adherent toepithelial cells (16HBE140) stained with:

FIG. 7A: Neisseria meningitidis L4 galE adherent to epithelial cells(16HBE140) stained with MAb A4 (anti-mouse TRITC-red).

FIG. 7B: Neisseria meningitidis L4 galE adherent to epithelial cells(16HBE140) stained with anti-cap B (anti-rabbit FITC-green).

FIG. 7C: Neisseria meningitidis L4 galE adherent to epithelial cells(16HBE140) stained with triple staining with MAb A4 (anti-mouseTRITC-red), anti-cap B (anti-rabbit FITC-green) and epithelial cellsstained DAPI (blue).

FIGS. 7D and 7E illustrate MAb B5 accesses inner core LPS epitopes inNeisseria meningitidis L3 MC58 (magnification ×2400).

FIG. 7D: Neisseria meningitidis L3 MC58 adherent to HUVECs stained withMAb B5 (antimouse TRITC-red)

FIG. 7E: Neisseria meningitidis L3 MC58 adherent to HUVECs stained withMAb B5 anti-cap B (anti-rabbit FITC-green) using confocalimmunofluorescence microscopy.

FIG. 8 illustrates conservation of the LPS epitope across Neisseriameningitidis serogroups.

FIG. 9 illustrates the strategy for the Example 2.

FIG. 10A illustrates ELISA titres of antibodies to L3 galE LPS (IgG) inpaired sera taken early and late from children with invasivemeningococcal disease.

FIG. 10B illustrates mean % phagocytosis of Neisseria meningitidis MC58with paired sera taken early and late from children with invasivemeningococcal disease with human peripheral blood mononuclear cells andhuman complement.

FIG. 11A illustrates mean % phagocytosis of Neisseria meningitidis MC58with MAb B5 pre-incubated with increasing concentrations of either (i)B5 reactive or (ii) B5 non-reactive galE LPS with human peripheral bloodpolymorphonuclear cells and human complement.

FIG. 11B illustrates mean % phagocytosis of pair of Neisseriameningitidis wild-type isogenic strains (Neisseria meningitidis BZ157)that are either MAb B5 reactive or B5 nonreactive with MAb B5 as theopsonin with human peripheral blood mononuclear cells and humancomplement.

FIG. 11C illustrates mean % phagocytosis of fluorescent latex beadscoated with either purified LPS from L3 galE mutant (10 μg/ml) oruncoated, in the presence of MAb B5 or final buffer, with humanperipheral blood mononuclear cells and human complement.

FIG. 12 illustrates mean % survival of Neisseria meningitidis galEmutant in the presence and absence of MAb B5 against two-fold serialdilutions of human pooled serum starting at 40% as detected using aserum bactericidal assay (see methods).

FIG. 13 illustrates geometric mean bacteremia in the blood of groups of5 day old infant rats 24 h post-infection with 1×10⁸ cfu/ml galE mutantgiven simultaneously with: (i) no antibody; (ii) MAb B5 (10 μg dose);(iii) MAb B5 (100 μg dose); or (iv) MAb 735, a positive controlanti-capsular antibody (2 μg dose).

FIG. 14 illustrates a Western blot showing purified LPS from Neisseriameningitidis MC58 and galE mutant probed with MAb B5 (ascites fluid1:2000) detected using anti-mouse IgG alkaline phosphatase and BCIP/NBTsubstrate.

FIG. 15A illustrates a FACS profile showing surface labeling of liveNeisseria meningitidis MC58 (5×10⁸ org./ml) with MAb B5 (culturesupernatant 1:50) detected using anti-mouse IgG (FITC labelled).

FIG. 15B illustrates a FACS profile comparing surface labeling of liveNeisseria meningitidis galE mutant (5×10⁸ org./ml) with MAb B5 (culturesupernatant 1:50) detected using anti-mouse IgG (FITC labeled).

DETAILED DESCRIPTION

The principal immunogenic component for Neisseria meningitidis strainsis preferably a single immunogenic component found in at least 50% ofNeisseria meningitidis strains, i.e. in the majority of naturallyoccurring Neisseria meningitidis strains. The principal immunogeniccomponent forms a candidate vaccine immunogen. Preferably theimmunogenic component of the vaccine of the present invention is any oneelement or structure of Neisseria meningitidis or other species ofNeisseria capable of provoking an immune response, either alone or incombination with another agent such as a carrier. Preferably theprincipal immunogenic component comprises of or consists of an epitopewhich is a part or all of the inner core structure of the Neisseriameningitidis LPS. The immunogenic component may also be derived fromthis inner core, be a synthetic version of the inner core, or be afunctional equivalent thereof such as a peptide mimic. The inner coreLPS structure of Neisseria meningitidis is generally defined as thatshown in FIGS. 1A and 1B and as outlined in the figure legends. Theimmunogenic component is suitably one which elicits an immune responsein the presence and in the absence of outer core LPS.

The principal immunogenic component is conserved in at least 50% ofNeisseria strains within the species, preferably at least 60%, and morepreferably at least 70%. Reactivity with 100% strains is an idealizedtarget, and so the immunogenic component typically recognizes at most95%, or 85% of the strains. Conservation is suitably assessedfunctionally, in terms of antibody cross-reactivity. We prefer that theimmunogenic component is present in at least 50% of serogroup B strains,preferably at least 60%, more preferably at least 70%, even morepreferably at least 76%. Suitably, assessment of the cross reactivity ofthe immunogenic component is made using a representative collection ofstrains, such those outlined in Maiden (Maiden M. C. J., et al., 1998.P.N.A.S. 195, 3140-3145).

Preferably the principal immunogenic component is found in the Neisseriameningitidis immunotype L3, and preferably it is not in L2. Morespecifically, we prefer that the immunogen is found in the immunotypesL1, L3, L7, L8 and L9, but not in L2, L4, L5 or L6. In other words, weprefer that the immunogen, notably the principal immunogenic component,generates antibodies which are reactive with at least the L3 immunotype,and usually the L1, L3, L7, L8 and L9 immunotypes, but not with L2, andusually not the L2, L4, L5 and L6 immunotypes. There are conformationaldifferences forced on the inner core of the L2 and L3 immunotypes bydifferent arrangements at HepII, namely the PEtn moiety at the6-position in L2 or at the 3-position in L3, and the Glc residue at the3-position in L2. Currently we do not envisage the possibility of asingle epitope for both L2 and L3 immunotypes. In other words, withoutdismissing the possibility of a single epitope, the present invention isexpected to require different immunogens to elicit antibodies for L2 andL3.

Preferably the principal immunogenic component is a conserved epitope onthe LPS inner core recognized by an antibody termed B5 herein. Thepreferred epitope of the invention is thus any epitope recognized by theB5 antibody.

Preferably the immunogenic component is a conserved epitope on the LPSinner core defined by the presence of a phosphoethanolamine moiety(PEtn) linked to the 3-position of HepII, the β-chain heptose, of theinner core, or is a functional equivalent thereof. In this respect wherethe context permits, HepI and HepII refer to the heptose residues of theinner core oligosaccharide which respectively are proximal and distal tothe lipid A moiety of the neisserial LPS, without being necessarily tiedto the general formula given above.

Preferably this epitope comprises a glucose residue on HepI, the α-chainheptose residue. While this glucose is not necessary for B5 biding, itis required for optimal recognition.

The principal immunogenic component of the present invention ispreferably an epitope on the LPS inner core which comprises an N-acetylglucosamine on HepII. The presence of N-acetyl glucosamine is requiredfor optimal recognition by B5.

Preferably the principal immunogenic component comprises both theN-acetyl glucosamine on HepII and a glucose residue on HepI.

The immunogenic component of the present invention is typically onlylimited by the requirement for a phosphoethanolamine moiety (PEtn)linked to the 3-position of HepII of the inner core, which is requiredfor B5 reactivity. The structure of the inner core may be modified,replaced, or removed, as necessary, to the extent that these are notneeded. Similarly, any outer core structures may be modified or deleted,to the extent that structural elements are not needed. There is norequirement for the immunogenic component to lack the outer coreportion, or equivalent, of the LPS. The immunogenic component maycomprise outer core elements having a galactose component, for examplethe terminal galactose residue of the lacto-N-neotetraose. In onesuitable embodiment, the immunogenic component is derived from LPS andis free from other cellular material. Alternatively, cellular materialmay be present, and can take the form of live or killed bacteria.

In a related aspect, the vaccine of this invention has an immunogenicepitope recognized by an antibody to a galE mutant of Neisseriameningitidis.

In a further embodiment the vaccine suitably comprises furtherimmunogenic elements from the inner core with an aim to achieving up to100% coverage. Preferably the vaccine comprises only a limited number(4-6, or less) of immunogenic elements, more preferably only thoseglycoforms which are representative of all possible PEtn positions onHepII, the β-chain heptose, of the inner core, i.e. wherein PEtn is atthe 3-position, exocyclic (6-position or 7-position) or absent, with orwithout an α-1-3 linked glucose at HepII, or a combination thereof. Thepresence of PEtn substituent is not required for the generation ofantibodies by an immunogenic component of this invention.

Moreover, as detailed herein, the epitopes of this invention areimmunogenic and accessible, and thus can be used to develop an effectivevaccine. Furthermore, as detailed herein, a vaccine containing only alimited number of glycoforms (representing all the possible PEtnpositions on HepII, namely position 3-, or 6- or 7-, or none, andcombinations thereof), is able to effectively provide protection againstthe diverse range of meningococcal isolates causing invasive disease.

Accordingly the vaccine of the present invention preferably comprises anepitope which is defined by the presence of a phosphoethanolamine moiety(PEtn) linked to the 3-position of HepII of the inner core, andadditionally comprises an epitope defined by the presence of PEtn on the6-position of HepII of the inner core, and/or an epitope defined by PEtnon the 7-position of HepII of the inner core, or wherein there is noadditional PEtn addition. Preferably the vaccine contains onlyimmunogenic components which are these inner core glycoform variants.

The B5 antibody of the present invention also recognizes the inner corestructures of Neisseria gonorrhoeae and Neisseria lactamica. As such,the invention extends to any Neisseria species, and any reference toNeisseria meningitidis can, as appropriate, be extended to otherNeisseria species, preferably Neisseria meningitidis, Neisseriagonorrhoeae, and Neisseria lactamica, most preferably Neisseriameningitidis. The invention also extends to immunogenic components inother Neisseria species which are related to those identified inNeisseria meningitidis, either by function, antibody reactivity orstructure. The invention is not limited to pathogenic strains ofNeisseria. The vaccine of this invention can be derived from a commensalstrain of Neisseria, especially a strain of Neisseria lactamica. Thespecies Neisseria lactamica is typically strongly immunogenic, andtherefore we prefer that the LPS inner core immunogenic component isderived from this species.

The vaccine may thus be homologous or heterologous, and thus founded onan immunogenic component from the target micro-organism, homologous, orfrom a different micro-organism, heterologous. The micro-organism can benaturally occurring or not, such as can be produced by recombinanttechniques. In particular, the micro-organism can be engineered tomodify the epitope or to modify other components.

In a further aspect of the invention we have determined that a secondmonoclonal antibody, herein termed A4, is able to react with inner coreepitopes of nearly all of the Neisseria meningitidis strains which donot react with the B5 antibody. Thus, of the 100 Neisseria meningitidisstrains tested, 30% were not reactive with B5 and were found to lack aPEtn moiety at the 3-position of HepII. Of these 30 strains, 27 werereactive with A4. Accordingly, a vaccine comprising only 2 inner coreepitopes, corresponding to those epitopes defined by cross reactivitywith A4 and B5, provides 97% coverage of a representative collection ofNeisseria meningitidis strains, preferably as assessed by using thecollection of strains as outlined in Maiden et al. [supra]. A preferredepitope of the invention is thus also any epitope recognized by the A4antibody.

A hybridoma producing the monoclonal antibody A4, designated hybridomaNmL4galEA4, has been deposited under the Budapest Treaty on 26 Sep. 2000with the International Depositary Authority of Canada in Winnipeg,Canada, and given the Accession Number IDAC 260900-2.

The present invention thus also relates to a vaccine comprising a fewimmunogenic components, wherein at least 70% of Neisseria meningitidisstrains of the species possess at least one of the immunogeniccomponents, preferably 80%, preferably 90%, and most preferably 97%. Inthis way the vaccine can give protective coverage against Neisseriainfection in 70%, preferably 80%, 90% or even 97% or more of cases.

A few immunogenic components suitably means at least two immunogeniccomponents, preferably only 2. More generally the few componentscomprise 2 to 6 components, such as 2, 3, 4, 5, or 6 components, moresuitably 2, 3, or 4 components. Preferably the immunogenic componentsare a few glycoforms of the inner core, representative of all naturalNeisseria meningitidis strains. In this way, a vaccine containing alimited number of glycoforms can give approaching 100% coverage ofNeisseria meningitidis strains.

A representation of the 3D structures of the LPS inner core having aPEtn moiety at the 3-position, 6-position or absent at HepII are shownin FIGS. 3A-3C. Accordingly, the present invention also extends toimmunogenic elements which have the same or similar structures to theseinner core structures, as defined by their 3D geometry and to antibodiescapable of interacting with such structures, either as assessed invitro, in vivo or in silico.

The immunogenic elements of the invention are preferably those shown toelicit antibodies having opsonic and bactericidal activity, and shown togenerate antibodies which confer passive protection in in vivo models.

The invention also extends to use of any immunogenic element as definedabove in the preparation of a medicament for the prevention, treatmentor diagnosis of Neisseria infection.

The candidate vaccine immunogens of the present invention may be suitedfor the prevention of all Neisseria infections. However, a vaccine forthe treatment of Neisseria meningitidis is preferred, with a vaccine forgroup B strains especially preferred.

Preferably the immunogenic element of the vaccine is accessible in thepresence of bacterial capsule. Accordingly, antibodies generated by anindividual who is vaccinated will be able to access the same epitope oninvading strains of Neisseria, and thus protect the individual frominfection. Antibodies given directly to a patient for treatment also arethus able to directly access the target Neisseria strains.

Preferably the vaccine of the present invention comprises epitopes whichare capable of stimulating antibodies which are opsonic. We furtherprefer that these antibodies are capable of binding to wild typeNeisseria strains to confer protection against infection and which arebactericidal.

The present invention also provides a method for treating pathogenicNeisseria. The method employs one or a few immunogenic components whichgive rise to effective antibodies and which rely on an inner coreepitope for stimulating the immune response. The immune response isordinarily B cell mediated, but we can include T cell mediated immunity.The antibodies generated by the vaccine of this invention bind to innercore elements of the pathogenic target bacterium.

Diseases caused by Neisseria meningitidis include principallymeningitis, septicaemia and pneumonia, and the prevention and treatmentof these diseases is especially preferred in the present invention.Diseases caused by Neisseria gonorrhoeae include sexually transmitteddiseases such as urethritis, cervicitis, proctitis pharyngitis,salpingitis, epididymitis and bacteremia/arthritis. Additionally, theinvention extends to treatment and prevention of any other disease whichresults from Neisseria infection, especially to diseases in whichNeisseria infection could weaken the immune system such that anotherdisease or pathogen could be harmful to an individual. The treatment canbe preventative or curative.

The vaccine of the present invention is a formulation suitable for safedelivery to a subject, allowing the subject to develop an immuneresponse to future infection by Neisseria. Vaccines of the presentinvention are preferably formulated vaccines in which any of theimmunogenic components of the vaccine may be conjugated, and anysuitable agent for conjugation may be used. Conjugation enablesmodification of the presentation of the antigen, and may be achieved byconventional techniques. Examples of agents for conjugation includeproteins from homologous or heterologous species. In this way, theimmunogenic component of the present invention forms a saccharidepeptide conjugate. Preferably the peptide portion comprises a T cellactivating epitope.

The vaccines of the present invention may be delivered with an adjuvant,to enhance the immune response to the immunogenic components. Suitableadjuvants include aluminium salts, oils in combination with bacterialmacromolecules, liposomes, muramyl dipeptide, ISCOMS, bacterial toxinssuch as pertussis, cholera and those derived from E. coli and cytokinessuch as IL-1, IL-2 and IFNγ.

The vaccine of the invention may be delivered by suitable means, such asby oral delivery or parenteral administration, injection, nutraceuticalor other delivery means, and may be provided in any suitable deliveryform such as tablets, pills, capsules granules, solutions, suspensionsor emulsions. Suitably the vaccine components are prepared in the formof a sterile, isotonic solution.

The present invention also extends to the monoclonal antibodies derivedfrom the concepts and methodologies described herein, including but notlimited to B5 and A4, and use of these antibodies in the treatment ofNeisseria infection. The invention also relates to pharmaceuticalpreparations comprising such antibodies in combination withpharmaceutically acceptable carrier. Such preparations may be deliveredby any suitable means, such as those exemplified above for vaccinedelivery, and used in combination with other active agents or adjuvants.

The correct dosage of the antibody or vaccine will vary according to theparticular formulation, mode of application, and the particular hostbeing treated. Factors such as age, body weight, sex, diet, and time ofadministration, rate of excretion, condition of the host, drugcombinations, and reaction sensitivities are suitably to be taken intoaccount.

The antibodies and vaccine compositions of the present invention may beused with other drugs to provide combination treatments. The other drugsmay form part of the same composition, or be provided as a separatecomposition for administration at the same time or a different time.

In addition to the antibodies themselves, the invention also relates tothe hybridomas which produce such antibodies.

Antibodies against the immunogenic components of the invention may begenerated by administering the immunogenic components to an animal,preferably a non-human animal using standard protocols. For thepreparation of monoclonal antibodies, any suitable techniques can beused. Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce appropriate single chainantibodies. Moreover, transgenic mice or other organisms or animal maybe used to express humanized antibodies immunospecific to theimmunogenic components of the invention.

Alternatively, other methods, for example phage display technology maybe used to select antibody genes for proteins with binding activitiestowards immunogenic components of the present invention.

Antibodies of the invention may be either monoclonal or polyclonalantibodies, as appropriate.

The present invention also relates to a method for the prevention ofNeisseria infection, the method comprising administering to a subject inneed of such treatment an effective amount of a vaccine as describedabove. Preferably the administration is adequate to produce a longlasting antibody and/or T cell immune response to protect the subjectfrom infection, particularly Neisseria meningitidis infection.

The invention also relates to a method for the treatment of Neisseriainfection, the method comprising administering to a subject in need ofsuch treatment an effective amount of an antibody to the Neisseriameningitidis inner core. Preferably, the antibody is B5 or A4, or anantibody which recognizes the same epitope as B5 or A4, or an antibodyderived from the concepts and methodologies herein described, or is acombination thereof.

Moreover, the methods of the invention may be extended to identificationof epitopes in any bacterial strain. Epitopes so identified may betested both for accessibility, conservation across the population andfunctional activity, using methods as outlined in the attached Examples.The present invention thus additionally relates to a method for theidentification of an immunogenic element, comprising raising an antibodyto a bacterial structure, preferably bacterial LPS structure, morepreferably a bacterial inner core LPS structure, and testing the epitoperecognized by the antibody for accessibility to antibody in the wildtype strain optionally also comprising testing the epitope forconservation across the bacterial population and testing for functionalactivity to the epitope in vivo.

Preferably the bacterial species are Neisseria species, preferablyNeisseria meningitidis, Neisseria gonorrhoeae or Neisseria lactamica.

Specifically, the present invention provides a method to generateantibodies to the inner core of Neisseria meningitidis. For the firsttime it has been possible to screen a population of Neisseriameningitidis strains to identify whole population features which areindependent of immunotype.

Accordingly, the present invention also relates to a method for theidentification of immunogenic epitopes of Neisseria meningitidis, themethod comprising the steps of:

1. generating antibodies to the inner core of Neisseria meningitidis, byinoculation of host organism with a galE mutant strain of Neisseriameningitidis, and

2. testing such antibodies against a wild type Neisseria meningitidisstrain to identify those antibodies which are reactive, and for whichthe epitopes are therefore accessible.

The potential utility of epitopes so identified may be further assessedby screening antibodies which react with the inner core of Neisseriameningitidis galE strain against a panel of strains which arerepresentative of strain diversity. Preferably the strain panel isselected using an approach based upon a population analysis. Epitopes soidentified may then be tested in functional assays, as outlined inExample 3.

In particular the invention extends to a method for the analysis ofantibody binding to bacteria wherein natural isolates of bacteria arestudied when grown on and adherent to tissue cultured cells, such asHUVECs. This assay provides a monolayer of cells to which the bacteriaadhere in a biologically relevant environment. Previous attempts usingNeisseria, for example, directly adherent to gelatin- or MATRIGEL-coatedcoverslips resulted in low numbers of adherent bacteria after repeatedwashings and high non-specific background staining. In particular weprefer that the antibody binding is analyzed using confocal microscopy.

This method also identifies antibodies suitable for therapeutic use, andthe invention extends to such antibodies.

Moreover, key biosynthetic genes for each step in LPS synthesis havebeen identified (Preston et al., 1996. Crit. Rev. Microbiol. 22:139-180) and this allows the construction of a series of mutants fromwhich LPS glycoforms of varying size and complexities can be madeavailable to facilitate the identification of conserved epitopes (vander Ley et al., 1997. FEMS Microbiol. Letter 146: 247-253; Jennings etal., 1993, Mol. Microbiol. 361-369; Jennings et al., 1995. Microb.Pathog. 19: 391-407; van der Ley et al., 1996, Mol. Microbiol. 19:1117-1125).

The present invention also relates to the gene found in Neisseriameningitidis which is involved in PEtn substitution at the 3-position onHepII, and to genes related in structure and function. As yet no geneshave been identified in any bacteria that are involved in addition ofPEtn to LPS structures. Using B5, specific for an inner core LPS epitopecontaining a PEtn, we have identified a putative LPS phosphoethanolaminetransferase gene (designated hypo3) in Neisseria meningitidis. Hypo3 wasnamed arbitrarily by us, as it is the 3rd reading frame in a fragment ofDNA selected by experimentation, from the MC58 genome sequence. Theoriginal hypo3 is therefore from MC58. This ORF is called NMB2010 in theTIGR data base (MC58 genome sequence) and although designated as aprotein of unknown function, they classify it as a “YhbX/YhjW/YijP/YjdBfamily protein”. This indicates that homologues have been inferred inother organisms but they do not know the function of them. The homologuein the serogroup A sequence at the Sanger Centre is designated NMA0431,although this gene is smaller than hypo3. Hypo3 is involved in PEtnsubstitution at the 3-position at HepII. Furthermore, the presence ofthe complete gene is required for the expression of the B5 reactivephenotype in Neisseria meningitidis as well as other pathogenic andcommensal Neisseria species.

The identification of the gene allows mutants to be created which areisogenic apart from hypo3, and which differ only in the presence orabsence of PEtn at the 3-position of HepII in the LPS inner core. Suchstrains can be used in comparative studies. Moreover, strainsappropriate for vaccine production can be engineered so that theycomprise the preferred PEtn structure at the 3-position, or engineeredso that this PEtn cannot be present.

Accordingly, the invention relates to use of the hypo3 gene, orhomologue thereof, in the production of a Neisseria strain for theassessment, treatment or prevention of Neisseria infection. Thehomologue may have 60%, 70%, 80%, 90% or more homology or identity tohypo3, as assessed at the DNA level. Use of the gene includes themethods outlined above, for preparing genetically modified strains forvaccination, isolation of appropriate epitopes and generation of strainsfor comparative studies. More generally, we envisage the identificationand use of any gene which plays a role in the biosynthetic pathway, andwhich has an effect on the conservation, accessibility or function ofthe immunogen.

EXAMPLES Example 1 Identification of Immunogenic Epitopes in Neisseriameningitidis Introduction

We investigated the conservation and antibody accessibility of innercore epitopes of Neisseria meningitidis lipopolysaccharide (LPS) becauseof their potential as vaccine candidates. An IgG3 murine monoclonalantibody (MAb), designated MAb B5, was obtained by immunizing mice witha galE mutant of Neisseria meningitidis H44/76 (B.15.P.1.7.16 immunotypeL3). We have shown that MAb B5 can bind to the core LPS of wild-typeencapsulated MC58 (B.15.PI.7.16 immunotype L3) organisms in vitro andex-vivo. An inner core structure recognized by MAb B5 is conserved andaccessible in 26/34 (76%) of Group B and 78/112 (70%) of Groups A, C, W,X, Y, and Z strains. Neisseria meningitidis strains which possess thisepitope are immunotypes in which phosphoethanolamine (PEtn) is linked tothe 3-position of the β-chain heptose (HepII) of the inner core. Incontrast, Neisseria meningitidis strains lacking reactivity with MAb B5have an alternative core structure in which PEtn is linked to anexocyclic position (i.e. position 6 or 7) of HepII (immunotypes L2, L4and L6) or is absent (immunotype L5). We conclude that MAb B5 definesone or more of the major inner core glycoforms of Neisseria meningitidisLPS.

These findings encourage the possibility that immunogens capable ofeliciting functional antibodies specific to inner core structures couldbe the basis of a vaccine against invasive infections caused byNeisseria meningitidis.

In summary, we report that a monoclonal antibody, designated B5, hasidentified a cross-reacting epitope on the LPS of the majority ofnaturally occurring, but genetically diverse strains of Neisseriameningitidis. Critical to the epitope of strains recognized by themonoclonal antibody B5 is a phosphoethanolamine (PEtn) on the 3-positionof the β-chain heptose (HepII) (FIG. 1A). In contrast, all Neisseriameningitidis strains lacking reactivity with MAb B5 are immunotypescharacterized by the absence of PEtn substitution or by PEtnsubstitution at an exocyclic position (i.e. position 6 or 7) of HepII(FIG. 1B). Thus, a limited repertoire of inner core LPS variants isfound among natural isolates of Neisseria meningitidis strains and thesefindings encourage the possibility that a vaccine might be developedcontaining a few glycoforms representative of all natural Neisseriameningitidis strains.

Materials and Methods Bacterial Strains

The Neisseria meningitidis strains MC58 and H44/76 (both P:15:P1.7.16immunotype L3) have been described previously (Virji, M., et al., 1991.Mol. Microbiol. 5: 1831-1841; Holten, E. 1979. J. Clin. Microbiol. 9:186-188). Derivatives of MC58 and H44/76 with defined alterations in LPSwere obtained by inactivating the genes, galE (Jennings, M. P., et al.,1993. Mol. Microbiol. 10: 361-369), Isi (Jennings, M. P., et al., 1995.Microb. Pathog. 19: 391-407), IgtA, IgtB (Jennings, M. P., et al., 1995.Mol. Microbiol. 18: 729-740), rfaC (Stoiljkovic, I., et al., 1997. FEMSMicrobiol Lett. 151: 41-49), icsA and icsB (van der Ley, P., et al.,1997. FEMS Microbiol. Lett. 146: 247-253) (Table 1). Other wild typeNeisseria meningitidis strains used in the study were from threecollections: 1) representatives of immunotypes L1-L12 (Poolman, J. T.,et al., FEMS Microbiol Lett. 13: 339-348); 2) global collection of 34representative Neisseria meningitidis Group B strains (Seiler, A., etal., 1996. Mol. Microbiol. 19: 841-856); 3) global collection of 100strains from 107 representative Neisseria meningitidis strains of allmajor serogroups (A, B, C, W, X, Y, Z) (Maiden, M. C. J., et al., 1998PNAS 95: 3140-3145).

Capsule deficient and galE mutants were constructed in six Neisseriameningitidis Group B strains obtained from the collection as describedin (Seiler, A., et al., 1996. Mol. Microbiol. 19: 841-856) (Table 1).Other related Neisseria strains studied included 10 strains of Neisseriagonorrhoeae and commensal strains lactamica (8 strains), polysaccharea(1 strain), mucosa (1 strain), cinerea (1 strain), elongata (1 strain),sicca (1 strain) and subflava (1 strain). Other Gram negative organismsincluded: Haemophilus influenzae type b (7 strains), Haemophilus somnus(1 strain), non-typable Haemophilus influenzae (8 strains), Escherichiacoli (1 main) and Salmonella typhimurium (1 strain) and its isogenic LPSmutants (rfaC, rfaP, and rfaI) (Table 1).

Bacterial Culture In Vitro

All strains were grown overnight at 37° C. on standard BHI medium base(Oxoid) in an atmosphere of 5% CO₂

Bacterial Culture In Vivo Using the Chick Embryo Model

To determine the accessibility of inner core epitopes of Neisseriameningitidis grown in vivo the chick embryo model was used (Buddingh, G.J., and A. Polk. 1937. Science 86: 20-21; Buddingh, G. J., and A. Polk.1939. J. Exp. Med. 70: 485-498; Schroten, H., et al., 1995. Pediar.Grenzgeb. 34: 319-324). The method was modified using an inoculum of 10⁴and 10⁵ Neisseria meningitidis organisms in a final volume of 0.1 ml, toinfect the chorio-allantoic fluid of 10 day old Pure Sussex chick eggs(obtained from the Poultry Unit Institute of Animal Health, Compton,Berks). After overnight incubation (37° C.) the allantoic fluid (approx.3-5 mls) was removed from the eggs and the bacteria recovered aftercentrifugation at 350×g for 15 minutes. The organisms were washed insterile phosphate buffered saline (PBS) and stored in Greaves solution(5% BSA, 5% Sodium Glutamate, 10% Glycerol) at −70° C.

LPS Extraction

LPS samples were obtained from an overnight growth of Neisseriameningitidis plated on 5 BHI plates from which the organisms werescraped and suspended in 30 ml 0.05% phenol in PBS and incubated at roomtemperature for 30 minutes. Alternatively, batch cultures were preparedin fermenters using bacteria from an overnight growth (6 plates) in 50ml DIFCO BACTO Todd Hewitt broth (DIFCO) to inoculate 2.5 L of the samemedium. For insertion mutant strains, the medium contained 50 μg/mlkanamycin. Following incubation at 37° C. for 6-8 h the culture wasinoculated into 60 L of BACTO Todd Hewitt broth in a New BrunswickScientific 1F-75 fermenter. After overnight growth (17 h at 37° C.), theculture was killed by addition of phenol (1%), and chilled to 15° C. andthe bacteria were harvested by centrifugation (13,000 g for 20 min)(Wakarchuk W., et al., 1996. J. Biol. Chem. 271: 19166-19173). In eithercase, the crude LPS were extracted from the bacterial pellet using thestandard hot phenol-water method (Westphal, O., and J. K. Jann. 1965.Meth. Carbohydr. Chem. 5: 83-91) and purified from the aqueous phase byrepeated ultracentrifugation (105,000×g, 4° C., 2×5 h) (Masoud, H., etal., 1997. Biochemistry 36: 2091-2103).

Tricine Gels

Equivalent amounts of whole-cell lysates of Neisseria meningitidisstrains or purified LPS were boiled in dissociation buffer and separatedon standard tricine gels (30 mA for 18 h) (Lesse, A. J., et al., 1990.J. Immunol. Methods. 126: 109-117). Gels were fixed and silver-stainedas per manufacturer's instructions (BioRad). To determine the presenceof sialic acid, whole cell lysates were incubated with 2.5 μlneuraminidase at 37° C. for 18-20 h (4 U/ml Boehringer 1585886) and thenwith 5 μl proteinase K at 60° C. for 2-3 h to remove proteins(Boehringer 1373196) prior to separation on tricine gels (16.5%).

Characterization of LPS from MAb 85 Negative Strains

LPS from wild-type and galE, cap-mutant MAb B5 negative strains wereO-deacylated with anhydrous hydrazine as described previously (Masoud,H., et al., 1997. Biochemistry 36: 2091-2103). O-deacylated LPS wasanalyzed by electrospray mass spectrometry (ES-MS) in the negative ionmode on a VG Quattro (Fisons Instruments) or API 300(Perkin-Elmer/Sciex) triple quadruple mass spectrometer. Samples weredissolved in water which was diluted by 50% with acetonitrile:water:methanol: 1% ammonia (4:4:1:1) and the mixture was enhanced bydirect infusion at 4 μl/min. Deacylated and dephosphorylated LPS (L8 odAHF) was prepared according to the following procedure. LPS (160 mg) wastreated with anhydrous hydrazine (1.5 ml) with stirring at 37° C. for 30minutes. The reaction was cooled (0° C.), cold acetone (−70° C., 50 ml)was added gradually to destroy excess hydrazine, and precipitatedO-deacylated LPS (L8 odA) was obtained by centrifugation. L8 odA waswashed twice with cold acetone, and redissolved in water andlyophilized. The structure of L8 odA was confirmed by negative ion ES-MSbefore proceeding to dephosphorylation. L8 odA was dephosphorylated bytreatment with 48% aqueous hydrogen fluoride (10 ml) at 0° C. for 48 h.The product was dialyzed against water, and the O-deacylated,dephosphorylated LPS sample (L8 odA HF) was lyophilized (50 mg). Loss ofphosphate was confirmed by ES-MS.

Molecular Modeling

Molecular modeling of LPS epitopes was carried out as describedpreviously by Brisson, J. R., S. et al., 1997. Biochemistry 36:3278-3292). The starting geometry for all sugars was submitted to acomplete refinement of bond lengths, valence and torsion angles by usingthe molecular mechanics program MM3(92) (QPCE). All calculations wereperformed using the minimized co-ordinates for the methyl glycoside. Thephosphorus groups were generated from standard co-ordinates (ALCHEMY,Tripos software) and minimum energy conformations found in crystalstructures. Calculations were performed using the Metropolis Monte Carlo(MMC) method. All pendant groups were treated as invariant except forthe phosphorus groups which were allowed to rotate about the Cx-Ox andOx-P bonds. The starting angles for the oligosaccharide were taken fromthe minimum energy conformers calculated for each disaccharide unitpresent in the molecule. 24-dimensional MMC calculations of thehexasaccharides with or without PEtn groups attached were carried outwith 5000 macro moves.

The graphics were generated using the Schakal software (Egbert Keller,Kristallographischeslnstitut der Universitat, Freibury, Germany).

Antibodies Rabbit Polyclonal Antibody

We used a rabbit polyclonal antibody specific for Group B Neisseriameningitidis capsular polysaccharide obtained by immunizing a rabbit sixtimes sub-cutaneously with lysates of MC58 at 2-week intervals. Thefirst and second immunizations contained Freund's complete adjuvant andFreund's incomplete adjuvant respectively. Serum was obtained from bleed6. To increase specificity for the Group B capsular polysaccharide,rabbit polyclonal antibody (1 ml) was incubated overnight at 4° C. withethanol-fixed capsule-deficient MC58 (5×10⁹ org./ml). This pre-adsorbedpolyclonal antibody did not react with a capsule-deficient mutant ofMC58 using immunofluorescence microscopy.

Monoclonal Antibodies to Inner Core LPS

Murine monoclonal antibodies to H44/76 galE LPS were prepared bystandard methods. Briefly, 6-8 week old Balb/c mice were immunized threetimes intraperitoneally followed by one intravenous injection withformalin-killed galE mutant whole cells. Hybridomas were prepared byfusion of spleen cells with SP2/O—Ag 14 (Shulman, M., et al., 1978.Nature 276: 269-270) as described (Carlin, N. I., et al., 1986. J.Immunol. 137: 2361-2366). Putative hybridomas secreting galE specificantibodies were selected by ELISA employing purified LPS from L3 and itsgalE mutant, and L2. Ig class, subclass and light chain were determinedby using an isotyping kit (Amersham Canada Ltd, Oakville, Ontario).Clones were expanded in Balb/c mice following treatment with pristane togenerate ascitic fluid. Spent culture supernatant was collectedfollowing in vitro culture of hybridoma cell lines. Further testing ofgalE MAbs was carried out by screening against purified LPS fromNeisseria meningitidis L3 IgtA, IgtB, and IgtE mutant strains (FIG. 1A),and Salmonella typhimurium Ra and Re mutants. One of the MAbs, MAb B5(IgG₃), was selected for more detailed study.

Immunotyping Monoclonal Antibodies

To determine the immunotypes of Neisseria meningitidis strains studies,especially L2 and L4-L6, the following murine MAbs were used in dotblots and whole cell ELISA: MN42F12.32 (L2,5), MN4A8B2 (L3,7,9), MN4C1B(L4,6,9), MN40G11.7 (L6), MN3A8C (L5) (Scholten, R. J., et al., J. Med.Microbial. 41: 236-243).

Human Umbilical Vein Endothelial Cell (HUVEC) Assay

Cultured human umbilical vein endothelial cells (HUVECs) were preparedas described previously (Virji, M., et al., 1991. Microb. Pathog. 10:231-245) and were infected with strains of Neisseria meningitidis for 3h at 37° C. Neisseria meningitidis strains were grown either in vitro orin vivo using the chick embryo model (as described above). Theaccessibility of the inner core LPS epitopes of whole-cell Neisseriameningitidis to specific MAb B5 was determined using immunofluorescenceand confocal microscopy. Gelatin-coated glass coverslips coated withHUVECs were infected with wild-type Neisseria meningitidis as describedpreviously (Virji, M., et al., 1991. Mol. Microbial. 5: 1831-1841),except bacteria were fixed with 0.5% paraformaldehyde for 20 min insteadof methanol. For accessibility studies, coverslips were washed with PBS,blocked in 3% BSA-PBS and incubated with MAb B5 culture supernatant andpre-adsorbed polyclonal rabbit anti-capsular antibody. Binding ofantibody to wild-type Neisseria meningitidis strains was detected byanti-mouse IgG rhodamine (TRITC) (Dako) and anti-rabbit IgG fluorescein(FITC) (Sigma). HUVECs were stained using diaminophenylamine DAPI (1μg/ml) (Sigma). Mounted coverslips were viewed for immunofluorescenceusing appropriate filters (Zeiss Microscope with Fluorograbber, AdobePhotoshop or confocal microscope (Nikon Model).

ELISA Purified LPS ELISA

A solid phase indirect ELISA employing purified LPS was used todetermine the binding specificities of MAbs. NUNC MAXISORP plates werecoated overnight with 1.0 μg/well of purified LPS derived from wild typeand mutants. LPS (10 μg/ml) was diluted in 0.05M carbonate buffercontaining 0.02M MgCl₂, pH 9.8. Non-specific binding sites were blockedfor 1 h with 1% BSA-PBS (Sigma) and washed three times with PBS-TWEEN 20(0.05% v/v) (PBS-T). Plates were incubated for 1 h with MAb B5 culturesupernatant and washed three times in PBS-T. Primary antibody wasdetected using anti-mouse IgG-alkaline phosphatase (Sigma: CedarlaneLaboratories Ltd.) incubated for 1 h, washed three times in PBS-T, anddetected using p-nitrophenyl phosphate AP substrate system (Sigma:Kirkegaard & Perry Laboratories). The reaction was stopped after 1 hwith 50 μl 3M NaOH and absorbance determined at OD A_(405-410nm)(Dynatech EIA plate reader).

Inhibition ELISA

For inhibition ELISA studies, MAb B5 was incubated with purified LPSsamples prior to addition to L3 galE LPS coated plates and assayed asdescribed above.

Whole Cell ELISA

Whole cell (WC) ELISA was performed using heat-inactivated lysates ofNeisseria meningitidis organisms as described previously (Abdillahi, H.,and J. T. Poolman. 1988. J. Med. Microbial. 26: 177-180). NUNC MAXISORP96-well plates were coated with 100 μl bacterial suspension (OD of 0.1at A_(820nm)) overnight at 37° C., blocked with 1% BSA-PBS and identicalprotocol followed as for LPS ELISA.

Dot Blots

Bacterial suspensions prepared as above (2 μl) were applied to anitrocellulose filter (45 micron, Schleicher and Schueller) and allowedto air dry. The same procedure as described for WC ELISA was followedexcept the detection substrate was 5-bromo-4-chloro-3-indoylphosphate/nitroblue-tetrazolium (BCIP/NBT) (2 mg/ml; Sigma). The colorreaction was stopped after 30 min by several washes with PBS and blotswere air-dried.

Results

To investigate the potential of inner core LPS structures of Neisseriameningitidis as vaccines, we have studied the reactivity of an isotypeIgG₃ murine monoclonal antibody (MAb), designated B5, raised againstNeisseria meningitidis stain H44/76 immunotype L3 galE mutant. MAb B5was one of seven monoclonal antibodies to LPS inner core producedagainst Neisseria meningitidis immunotype L3 galE by standardimmunological methods (see Methods). Preliminary ELISA testing showed B5cross-reacted with LPS from L3 parent strain and with galE (IgtE), IgtAand IgtB mutants, but did not cross-react with Salmonella typhimurium Raor Re LPS.

In order to determine the specific inner core epitope recognized by MAbB5, various Neisseria meningitidis strains of known structure wereexamined in ELISA for cross reactivity (FIG. 2). The most significantfinding of this analysis was that Neisseria meningitidis immunotype L4LPS was not recognized by MAb B5. The only structural difference betweenimmunotypes L4 and L3 (which is recognized by MAb B5) is the position ofattachment of the PEtn group (FIGS. 3A-3C). In immunotype L3 LPS thePEtn is attached at the 3-position of HepII, whereas in immunotype L4LPS the PEtn is attached at the 6- or 7-position of HepII (FIGS. 3A and3B). Additionally, LPS from immunotype L2 and its galE mutant (in whichthe PEtn group is attached at the 6-position and a glucose residue ispresent at the 3-position of HepII) are not recognized by MAb B5.Immunotype L5, which has no PEtn in the inner core, is not recognized byB5, whereas immunotype L8 and its galE mutant which have PEtn at the3-position of HepII are recognized. These results suggest that MAb B5specifically recognizes PEtn when it is attached at the 3-position ofHepII.

In order to prove the essential inclusion of PEtn in the epitoperecognized by MAb B5, immunotype L8 O-deacylated (odA) LPS wasdephosphorylated (48% HF, 4° C. 48 h) (FIG. 3C). The absence of PEtnfollowing dephosphorylation was confirmed by ES-MS analysis. Asindicated in FIG. 4, dephosphorylation of L8 odA LPS abolishedreactivity to MAb B5. To further characterize the epitope recognized byMAb B5, several structurally defined genetic mutants of immunotype L3were screened for cross-reactivity (FIG. 4). The highly truncated LPS ofmutant strain icsB were only weakly recognized, while mutant strain icsALPS was not recognized by MAb B5. These results suggest that thepresence of glucose on the proximal heptose reside (HepI) is notabsolutely necessary for binding by B5 but is required for optimalrecognition (FIG. 1A). Furthermore, MAb B5 does not bind LPS in whichboth the glucose on the α-chain, HepI, and the N-acetylglucosamineresidue on the β-chain, HepII, are absent. This suggests that thepresence of N-acetylglucosamine is required to present the PEtn residuein the correct conformation for binding by MAb B5. Genetic modificationsthat produce severely truncated LPS glycoforms were also examined forreactivity with MAb B5. LPS from immunotype L3 Isi which has atrisaccharide of Hep-Kdo-Kdo attached to lipid A, and L3 PB4 which onlycontains the Kdo disaccharide and lipid A were not recognized by MAb B5(FIG. 4). Inhibition ELISA studies (data not shown) were in accord withthis result, thus confirming the specificity of MAb B5 to the PEtnmolecule linked at the 3-position of HepII.

To demonstrate the ability of MAb B5 to recognize this inner coreepitope in encapsulated strains, we devised an assay in which naturalisolates of Neisseria meningitidis were studied when they were grown onand became adherent to tissue cultured cells (HUVECs). Initially, thismethodology was developed using the fully encapsulated strain MC58. Theadvantages of using the HUVEC assay were that they provided a monolayerof endothelial cells to which the bacteria could adhere and that theyprovided a biologically relevant environment. Previous attempts usingNeisseria meningitidis directly adherent to gelatin- or MATRIGEL-coatedcoverslips resulted in low numbers of adherent bacteria after repeatedwashings and high nonspecific background staining.

Primary antibodies, MAb B5 and a polyclonal anti-capsular antibody weredetected by anti-mouse TRITC and anti-rabbit FITC respectively. Thisdemonstrated that an inner core LPS epitope of the fully encapsulatedstrain (MC58) was accessible to MAb B5 (FIG. 5A). Confocal microscopyshowed that MAb B5 and anti-capsular antibodies co-localized. Inaddition to this in vitro demonstration of accessibility of MAb B5 toinner core LPS, we also investigated organisms grown in vivo using thechick embryo model. Strain MC58 (10⁴ org./ml) was inoculated intochorioallantoic fluid of 10 day old chick embryos and harvested the nextday to provide ex-vivo organisms. The results of confocal microscopywere identical to those observed in vitro, that is MAb B5 andanti-capsular antibodies co-localized (FIG. 5B). This demonstrated thatthe inner core LPS epitopes were also accessible in vivo onwhole-encapsulated wild-type Neisseria meningitidis.

The observation of double staining of the inner core LPS epitope in thepresence of capsule is key to the concept of this approach and thereforea number of controls were used to confirm the validity finding. Theseincluded: (i) double staining a MAb B5 negative e.g. immunotype L4strain with MAb B5 and anti-capsular antibody. This resulted in noreactivity of MAb B5 on rhodamine filter but positive reactivity withanti-capsular antibody. This rules out a band passing effect during therecording of the pictures; (ii) single staining of encapsulated MAbB5positive strains with either MAb B5 alone or anti-capsular antibodyalone followed by staining with rhodamine or FITC, respectively. Whenviewed on the appropriate wavelength there was no cross-reactivityduring immunofluorescent staining nor any band-passing effect; (iii)double-staining of a MAb B5 positive or negative strain without capsulewith MAb B5 and anti-capsular antibody resulted in no capsular stainingbut either MAb B5 positive or negative reactivity when viewed on therhodamine filter. This excluded cross-reactivity during staining orband-passing effect resulting in artefactual inner core staining.

To survey the extent of MAb B5 reactivity with other Neisseriameningitidis strains, three collections were investigated.

i) 12 strains representative of LPS immunotypes L1-L12

ii) 34 Group B strains selected to represent genetically diverseisolates from many different countries obtained between the years1940-1988 (Seiler, A., et al., 1996. Mol. Microbiol. 19: 841-856)

iii) a global collection of 107 genetically diverse strains representingall capsular serogroups, also obtained from different countries from1940-1994 (Maiden, M. C. J., et al., 1998. PNAS 95: 3140-3145).

Of the 12 immunotypes, MAb B5 recognized the LPS of strains in which theinner core oligosaccharide has a PEtn linked to the 3-position of HepII(Table 2 and FIG. 1A). Thus, immunotypes L2, L4, L6 did not react withMAb B5, whereas immunotypes L1, L3, L7-L12 were recognized by MAb B5.This confirmed that the presence of PEtn in the 3 position of the HepIIis necessary to confer MAb B5 reactivity (FIG. 3A).

To investigate further the MAb B5 reactivity with other Group B strains,a collection of genetically diverse strains was studied (Seiler, A., etal., 1996. Mol. Microbiol. 19: 841-856). MAb B5 reactivity was detectedin 26/34 (76%) of Group B Neisseria meningitidis stains tested. Thisincluded representative strains of ET-5, ET-37, A4 and Lineage-3. Thisrepresents the most complete available collection of hyper-invasivelineages of Neisseria meningitidis Group B strains.

We obtained capsule-deficient and galE mutants from six of eight of theMAb B5 negative Group B strains (transformations were unsuccessful inthe other two strains). These were also negative with MAb B5 using dotblot, whole cell ELISA or immunofluorescence, with the exception of aBZ157 galE cap-mutant that had low level reactivity both byimmunofluorescence and dot blot. The MAb B5 strains were characterizedusing a battery of immunotyping MAbs. We determined the immunotype ofthe eight MAb B5 negative strains using combinations of the appropriateMAbs (see Methods) and dot blots of WC lysates (obtained from Peter vander Ley) (Table 3). In addition, structural fingerprinting of the innercore region of MAb B5 negative strains was performed by ES-MS onO-deacylated LPS from five of the respective capsule-deficient galEmutants (1000, NGE30, EG327, BZ157, NGH38) (Table 4). Strains 1000,NGE30, EG327 were non-typical by MAbs and LPS from these strains lackedPEtn on HepII of the inner core. BZ157, which corresponded to immunotypeL2 by MAbs contained PEtn in the inner core, and by analogy to L2, atthe 6/7 position of HepII (Table 3). NGH38 was immunotype L2, L5, andanalogous to L2 by structural analysis. Those strains that werenon-typable failed to react with MAbs that recognize L3,7,9, L6, L2,5,L4,6,9. However, 15/17 MAb B5 negative Neisseria meningitidis strains(all serogroups) were positive for L2, 5 and all MAb B5 positive strainswere positive for L3,7,9. No reaction with any immunotyping MAbs wasobserved with 8/32 MAb B5 negative strains and 24/68 of MAb B5 positivestrains.

To determine if the degree of sialylation of the LPS was a factor in theability of MAb B5 to recognize its inner-core epitope, MAb B5 negativestrains were examined by LPS gels. MAb B5 reactivity was unaffected byvarying the state of sialylation through exposure to neurominidase asdescribed in methods (FIGS. 6A and 6B). Furthermore, strain MC58, withwhich the MAb B5 reacted strongly, was found to be highly sialylated(FIG. 6B) and this was confirmed by ES-MS of purified O-deacylated LPS(data not shown). Therefore our data did not support a contribution ofsialylation to the lack of MAb B5 reactivity.

With respect to the other Neisseria species, MAb B5 also recognized theinner core LPS of five strains of Neisseria gonorrhoeae (F62, MS11,FA19, 179008, 150002) (two were negative) and (at least) two strains ofNeisseria lacramica (L19, L22). However, MAb B5 did not react with onestrain each of Neisseria polysaccharide (M7), Neisseria mucosa (F1),Neisseria cinerea (Griffiss, J. M., et al., 1987. Infect. Immun. 55:1792-1800), Neisseria elongata (Q29), Neisseria sicca (Q39) andNeisseria subflava (U37). Also MAb B5 did not react with Escherichiacoli (DH5 alpha), Salmonella typhimurium (LT2) or its isogenic LPSmutants (rfaC, rfaI, rfaP).

Finally, we investigated the reactivity of MAb B5 with 100 strains thatincluded representatives of serogroups A, B, C, W, X, Y and Z (Maiden,M. C. J., et al., 1998. PNAS 95: 3140-3145). Of these strains, 70% wereMAb B5 positive. Clustering according to genetic relatedness wasevident. For example, none of the MAb B5 negative stains were in the ET5complex. Among Group A strains, MAb B5 positive and negative stains alsofell into distinct clusters. For example, lineages I-III and lineage A4were positive and lineage IV-I was negative. This collection, togetherwith that described in (Seiler, A., et al., 1996. Mol. Microbiol. 19:841-856) represents the most complete set available for knownhyperinvasive lineages in all major serogroups of Neisseria meningitidisstrains.

Discussion and Conclusions

The pre-requisites for any candidate Neisseria meningitidis Group Bvaccine would be that it contains a highly conserved epitope(s) that isfound in all Group B stains and is accessible to antibodies in thepresence of capsule. Our approach has combined genetics, structuralanalysis and immunobiology to define candidate epitopes in inner coreLPS of Neisseria meningitidis Group B. This study uses murine MAb B5,isotype IgG3, which was raised to a genetically defined immunotype L3galE mutant in order to specifically target inner-core LPS epitopes. Theepitope(s) recognized by MAb B5 was defined by cross-reactivity studieswith purified LPS glycoforms of known structure. MAb B5 recognized allLPS glycoforms in which the PEtn is at the 3-position of HepII(immunotypes L1, L3, L7, L8, and L9) and failed to react withimmunotypes where PEtn is at the 6- or 7-position (L2, L4, and L6) orabsent from HepII (L5) (FIGS. 1A-1B). MAb B5 reacted with 70% Neisseriameningitidis strains tested from the two most complete sets of Neisseriameningitidis strains available word-wide (Seiler, A., et al., 1996. Mol.Microbiol. 19: 841-856, 35). Of these strains, 76% of Neisseriameningitidis Group B strains tested were positive with MAb B5 and 70% ofa collection that included all Neisseria meningitidis serogroups testedwere positive with MAb B5. Therefore, it may be envisaged that a vaccinecontaining a limited number of glycoforms, representing all the possiblePEtn positions (none, 3 and 6/7) on HepII on the inner core, would cover100% of Neisseria meningitidis Group B strains.

The LPS structures of MAb B5 negative strains were confirmed bystructural analysis. Two structural variants were recognized. Onevariant without PEtn in the inner core LPS (e.g. NGE30, EG327, 1000);and the other, with PEtn group of HepII (e.g. BZ157, NGH38) at the 6- or7-position instead of the 3-position.

With a view to developing inner core LPS epitopes as vaccine candidates,it is significant that there were no effects of the capsule on MAb B5accessibility, as shown by co-localization of the anti-capsule antibodyand MAb B5 in wild-type organisms (MC58) grown in vitro and in vivo byconfocal microscopy (FIGS. 5A and 5B). Nor did the presence or absenceof sialic acid have an effect since both MAb B5 positive and negativestrains had high sialylation states as shown by tricine gels (FIGS. 6Aand 6B) and confirmed by ES-MS (data not shown). There was no evidenceof phase variation in MAb B5 positive or negative strains in this study,with the exception of one strain (BZ157) which had a very low level ofMAb B5 positive strains in parent and galE mutant (0.06%) (data notshown). Structural analysis of LPS extracted from these two variants iscurrently under investigation.

Three-dimensional space filling models of the inner core LPS of L3 andL4 immunotypes show that the position of the PEtn, either 3- or6-position respectively, alters the accessibility and conformation ofPEtn in the inner core epitope (FIGS. 3A and 3B). The most strikingexample of the importance of PEtn for MAb B5 reactivity was observedwhen PEtn was removed from the immunotype L8 (MAb B5 positive) bytreatment with hydrogen fluoride (HF) which totally abolished MAb B5reactivity (FIG. 3C).

Previous studies with oligosaccharide conjugates in mice and rabbitshave demonstrated that PEtn is important in immunogenicity andfunctional activity of polyclonal antibodies (Verheul, A. F., et al.,1991. Med. Immun. 59: 843-851). These studies identified two sets ofpolyclonal antibodies. One set resulting from L1 and L3,7,9oligosaccharides had PEtn in the 3-position of HepII, were immunogenic,had opsonophagocytic (OP) and chemiluminescence in oxidative burstreaction, but had no serum bactericidal activity. The other set ofantibodies resulting from L2 conjugates (6- or 7-position or withoutPEtn at HepII) were poorly immunogenic and had greatly reduced OPactivity and chemiluminescence (Verheul. A. F., et al., 1991. Infect.Immun. 59: 843-851). Future studies will look at the safety andimmunogenicity of inner core LPS-conjugates (PEtn at 3-position of HepIIand alternative glycoforms) and the functional ability of thesepolyclonal antibodies in opsonic and serum bacterial assays, initiallyin mice and rabbits. Preliminary studies using MAb B5 in anopsonophagocytosis assays with Neisseria meningitidis strain MC58 anddonor human polymorphonuclear cells suggest MAb B5 is opsonic in thepresence of complement and that the uptake of Neisseria meningitidisbacteria correlates with an oxidative burst reaction within theneutrophil. MAbB5 does not appear to have any significant serumbactericidal activity with Neisseria meningitidis strain MC58, howeverthis is not unexpected in view of its isotype (IgG3). The functionalityof MAb B5 is currently under further investigation.

In conclusion, MAb B5 recognizes a conserved inner core epitope in whichthe PEtn is at the 3-position of HepII. This epitope was present in 76%Neisseria meningitidis Group B strains and 70% of all Neisseriameningitidis serogroups, and was accessible in the presence of capsule.A limited number of alternative glycoforms have been identified that arenot recognized by MAb B5 where the PEtn is either absent or at anexocyclic position of HepII. Therefore, a vaccine containing a limitednumber of glycoforms might give 100% coverage of all Neisseriameningitidis Group B strains.

TABLE 1 Bacterial strains. Relevant immunotype (bold) and Species Straingenotype(italics) Source/reference Neisseria meningitidis MC58 L3 CSFisolate Virji, M., et al., 1991. Mol Microbiol5: 1831-1841 H44/76 L3Holton, E. 1979. J Clin Microbiol 9: 186-188 MC58 galE Jennings, M. P.,et al., 1993. Mol. Microbiol. 10: 361-369 MC58 lsi1(rfaF) Jennings, M.P., et al., 1995 Microb. Pathog. 19: 391-407 MC58 lgtA Jennings, M. P.,et al., 1995. Mol. Microbiol. 18: 729-740 MC58 lgtB Jennings, M. P., etal., 1995. Mol. Microbiol. 18: 729-740 H44/76 rfaC Stolljokovic, I., etal., 1997. FEMS Microbial. Lett. 15 1: 41- 49 H44/76 icsA van der Ley,P., et al., 1997. FEMS Microbiol. Lett. 146: 247-253 H44/76 icsB van derLey, P., et al., 1997. FEMS Microbiol. Lett. 146: 247-253 126E; L1-L12Poolman, J. T., et al., 1982. 35E; H44/76; 89I; M981 RESPECTIVELY FEMSMicrobial. Lett. 13: 339- 348 M9926155; 892257; M978; 120M; 7880; 7889;3200 BZ157 L2 Seiler, A., et al., 1996. Mol. Microbiol. 19: 841-856BZ157 galE This study 1000 NT Seiler, A., et al., 1996. Mol. Microbiol.19: 841-856 1000 galE This study NGE30 NT Seiler, A., et al., 1996. Mol.Microbiol. 19: 841-856 NGE30 galE This study EG327 NT Seiler, A., etal., 1996. Mol. Microbiol. 19: 841-856 EG327 galE This study NGH38 L2, 5Seiler, A., et al., 1996. Mol. Microbiol. 19: 841-856 NGH38 galE Thisstudy EG328 NT Seiler, A., et al., 1996. Mol. Microbiol. 19: 841-856EG328 galE This study 3906; NGH15; Seiler, A., et al., 1996. Mol. BZ133;BZ83; EG329; Microbiol. 19: 841-856 SWZ107; BZ198; NGH41 NG4/88; 2970;BZ147; NGG40; NGH36; NG3/88; NGF26; NG6/88; NGH38; NGE28; BZ169; 528;DK353; BZ232 DK24; BZ159; BZ10; BZ163; NGP20 B40; Z4024; Z4081; Z2491;Z3524; (35) Z3906; Z5826; BZ10; BZ163; B6116/77; L93/4286; NG3/88;NG6/88; NGF26; NGE31; DK24; 3906; EG328; EG327; 1000; B534; A22; 71/94;860060; NGG40; NGE28; NGH41; 890326; 860800; NG4/88; E32; 44/76; 204/92;BZ8; SWZ107; NGH38; DK353; BZ232; E26; 400; BZ198; 91/40; NGH15; NGE30;50/94 88/03415; NGH36; BZ147; 297-0 Neisseria lactamica (L12. L13. L17,L18, L19, L20, L22) Brian Spratt & Noel Smith polysaccharea (P4)mucosa(M7), cinerea (F1), elongata (I8), sicca (Q29), subflava (U37)Neisseria gonorrhoeae: F62, MS11, FA19, FA1090, 179008, R. Goldstein150002, 15253 SN-4 Staffan Normavk P9-2 M. Virji Haemophilus influenzaetype b Hood, D. W., et al., 1996. Mol. Microbiol. 22: 951-964 Eagan;7004; Rd5B33; opsx 3Fe; E3Fi; E1B1 rfaF orfH. lpxA PLAK33 Steeghs, L, etal., 1998. Nature 392: 449-450. Haemophilus somnus 738 L1 J. RichardsNon-typable J. Eskola Haemophilus influenzae (NTHI): 54, 375, 477, 1003,1008, 1042, 1147, 1231 E. coli DH5α Neidardt, F. C., et al., (ed.), ASMPress. Salmonella typhimurium LT2 rfaC; rfa1; rfaP Schnaitman, C. A.,and F. D. Klena. 1993. 57: 655-682

TABLE 2 Reactivity of monoclonal antibody B5 with representativeNeisseria meningitidis strains of immunotypes L1-L12 determined by wholecell ELISA, dot blots of lysates, immunofluorescence and confocalmicroscopy. Serogroup: Serotype: Whole cell Immuno- Strain SerosubtypeImmunotype ELISA^(a)(OD_(A405 nm)) Dot Blot^(b) fluorescence^(c) 126EC:3:P1.5,2 L1 +1.8 +++ + 35E C:20:P1.1 L2 −<0.4 − − H44/76 B.15.P1.7,16L3 +1.3 +++ ++ 89I C:nt:P1.16 L4 −<0.4 − − M98I B:4:P1.— L5 −<0.4 +/− −M992 B:5:P1.7,1 L6 −<0.4 +/− − 6155 B:nt:P1.7,1 L7 +0.8 ++ + M978B:8:P1.7,1 L8 +1.9 +++ ++ 892257 B:4:P1,4 L8 +1.9 120M A:4:P1.10 L9 +1.8+++ + 7880 A:4:P1:6 L10 +2.2 +++ + 7889 A:4:P1.9 L11 +2.0 +++ ++ 3200A:4:P1.9 L12 +2.1 +++ ++ ^(a)Positive reactivity (OD_(A405) > 0.4) (+),negative reactivity (OD_(A405) < 0.4) (−) ^(b)Strongly positive (+++),positive (++), weakly positive (+/−), negative (−). ^(c)Stronglypositive (++), positive (+), negative (−).

TABLE 3 Correlation between reactivity with monoclonal antibody B5,immunotyping and location of phosphoethanolamine (PEtn) on HepII ofinner core. Position of PEtn on HepII Strain MAb B5 Immuno-type* O-3 O-6MC58 + L3,7 + − 1000 − NT − − NGE30 − NT − − EG327 − NT − − BZ157^(#) −L2,5 − + BZ157^(§) + L3,7 + − NGH38 − L2,5 − + Abbreviations: NT =non-typable *MN4A8B2 (L3,7,9); MN42F12.32 (L2,5); MN4C1B (L4,6,9);MN40G11.7 (L6) ^(#)BZ157 MAb B5 negative variant ^(§)BZ157 MAb B5positive variant

TABLE 4 Negative ion ES-MS data and proposed compositions ofO-deacylated LPS from galE capsule-deficient mutant Neisseriameningitidis MAb B5 negative strains. Observed Ions (m/z) Molecular Mass(Da) Strain (M − 2H)²⁻ (M − H)⁻ Observed Calculated Lipid A^(b) 10001213.0 2427.6 2427.7 2427.2 1075 1252.9 2507.8 2507.8 2507.2 1155 1314.52630.9 2603.9 2630.3 1278 NGH38 1293.8 2589.5 2589.3 2589.3 952 EG3271151.2 2304.4 2304.4 2304.1 952 NGE30 1132.1 — — 2265.1 1075 1396.12793.4 2793.7 2792.5 1075 1436.0 2873.7 2873.9 2872.5 1155 1498.0 2997.22997.1 2995.6 1278 BZ157 1274.6 2551.4 — 2550.3 1075 1314.8 2631.12631.2 2630.3 1155 1376.4 2754.4 2754.5 2753.4 1278 1457.5 2916.6 2916.62915.6 1278 Strain Proposed Composition^(a) 1000 2Glc, GlcNAc, 2Hep, 2Kdo, Lipid A 2Glc, GlcNAc, 2Hep, 2 Kdo, Lipid A 2Glc, GlcNAc, 2Hep, 2Kdo, Lipid A NGH38 3Glc, GlcNAc, 2Hep, PEtn, 2Kdo, Lipid A EG37 2Glc,GlcNAc, 2Hep, 2 Kdo, Lipid A NGE30 Glc, GlcNAc, 2Hep, 2Kdo, Lipid A3Glc, 2GlcNAc, 2Hep, 2 Kdo, Lipid A 3Glc, 2GlcNAc, 2Hep, 2 Kdo, Lipid A3Glc, 2GlcNAc, 2Hep, 2 Kdo, Lipid A BZ157 2Glc, GlcNAc, 2Hep, PEtn,2Kdo, Lipid A 2Glc, GlcNAc, 2Hep, PEtn, 2Kdo, Lipid A 2Glc, GlcNAc,2Hep, PEtn, 2Kdo, Lipid A 3Glc, GlcNAc, 2Hep, PEtn, 2Kdo, Lipid AAverage mass units were used for calculation of molecular weight basedon proposed composition as follows: Glc, 162.15; Hep, 192.17; GlcNAc,203.19; Kdo, 220.18; PEtn, 123.05. ^(a)Glc, glucose; GlcNAc,N-acetylglucosamine; PEtn, phosphoethanolamine; Hep, heptose; Kdo,3-deoxy-D-manno-octulosonic acid. ^(b)As determined by MS-MS analyses.

Example 2 Identification of Additional Inner Core Epitopes Introduction

Example 1 identifies an inner core LPS epitope that was accessible andconserved in 70% of a global collection of 104 Neisseria meningitidisstrains representative of all major serogroups (Plested et al., 1999,Infect. Immunity 67: 5417-5426). The epitope recognized by MAb B5 wasidentified in all LPS immunotypes with phosphoethanolamine (PEtn) in the3-position of β-chain heptose (HepII) of inner core LPS. Further workwas carried out to identify additional epitopes, with the aims outlinedin FIG. 4.

In Summary:

A series of twelve murine monoclonal antibodies (MAbs) were developed atNRC, by using a procedure described previously by us (Plested et al.,1999 Infect. Immunity 67: 5417-5426) except using formalin-fixedNeisseria meningitidis L4 (strain 891) galE whole-cells. The twelve MAbswere extensively screened by ELISA using purified LPS from Neisseriameningitidis mutants and wild-type strains and three MAbs B2 (IgG2b), A4(IgG2a), and A2 IgG2a were chosen for further investigation.Conservation of the inner core LPS epitope was assessed at Oxford usingwild-type whole-cell lysates of a global collection of 104 Neisseriameningitidis disease isolates (Maiden, M. C. J., et al., 1998. PNAS 95:3140-3145). Accessibility of the inner core LPS epitope was assessedusing immunofluorescence microscopy with ethanol-fixed Neisseriameningitidis whole-cells of wild type and mutants adherent to amonolayer epithelial cells (Plested et al., 1999).

Each of the three MAbs reacted with purified Neisseria meningitidis L4galE LPS by ELISA. Except for MAb B2 that had low reactivity withNeisseria meningitidis L4 LPS, none of the Neisseria meningitidis L4series of MAbs were able to the recognize wild-type L4 or L2 purifiedLPS by ELISA. None of the Neisseria meningitidis L4 MAbs recognizedNeisseria meningitidis wild-type L2 or L4 whole-cells byimmunofluorescence microscopy.

MAb B2 reacted with 15/32 Neisseria meningitidis MAb B5 negativeNeisseria meningitidis strains and 9/68 Neisseria meningitidis MAb B5positive Neisseria meningitidis strains by whole-cell dot blot analysis.MAb 2 reacted with L4 galE, L4 wild-type (very low reactivity) but notL3 galE, L2 galE (native) O-deacylated (odA)), L2 wild-type(native-odA), L5, L6 wild-type LPS.

MAb A2 recognized 28/32 Neisseria meningitidis MAb B5 negative Neisseriameningitidis strains and 20/68 Neisseria meningitidis MAb B5 positiveNeisseria meningitidis strains by whole-cell dot blot analysis. MAb A2reacted with L4 galE (native/odA), L2 galE (native) but not L3 galE, L2galE (odA), L2 wild-type (native/odA), L4, L5, L6 wild type LPS.

MAb A4 reacted with 29/32 Neisseria meningitidis MAb B5 negativeNeisseria meningitidis strains and 24/68 Neisseria meningitidis MAb B5positive Neisseria meningitidis strains by whole-cell lysate dot blotanalysis. MAb A4 reacted with L4 galE, L2 galE (native/odA), but not L3galE, L2 wild type, L4, L5, L6, L8 wild-type LPS.

Based on these results, MAb A4 (IgG2a) was chosen for further study asit demonstrated specificity for both L4 galE and L2 galE LPS by ELISAand recognized all except 3 Neisseria meningitidis B5 negative Neisseriameningitidis strains (BZ232 serogroup B; NGH38 serogroup B; F1576serogroup C). Together MAbs B5 and A4 were able to recognize 97/100Neisseria meningitidis isolates. Immunofluorescence microscopydemonstrated that MAb A4 was able to access the inner core epitope in anL4 galE mutant in the presence of capsule.

We have identified LPS inner-core epitopes with PEtn at the 3-positionof HepII (MAb B5) or not at the 3 position (MAb A4). There remain 3strains out of 100 (BZ232, NGH38 and F1576) which show no reactivitywith either MAb A4 or MAb B5. The structural basis for thisnon-reactivity is under investigation. Once all the variant glycoformsof the inner core are known, of which at least 3 have been identified,the rationale will exist for including epitopes, representative of allNeisseria meningitidis strains causing invasive disease, in a conjugatevaccine. This will be tested for proof in principle using studies inanimals before proceeding to human trials.

The following techniques were used:

(1) Murine MAb A4 (IgG2a) was raised to galE (89I, L4 immunotype) andselected on basis of reactivity in LPS ELISA & immunofluorescence (IF)microscopy.

(2) LPS ELISA (Plested et al., 2000. J. Immunol. Meth. 237: 73-84):Microtitre plates (Nunc) coated with purified (galE) LPS (10 μg/ml)overnight, were washed, blocked, incubated with MAb for 1 h, washed anddetected with anti-mouse IgG alkaline phosphatase and p-NPP(OD_(A405nm)).

(3) Immunoblotting using whole-cell lysates from 104 Neisseriameningitidis strains (Plested et al., 1999 IAI 67: 5417-5426). MAb A4was detected using anti-mouse IgG alkaline phosphatase and BCIP/NBT.

(4) Immunofluorescence microscopy: as before (Plested et al., 1999 IAI67: 5417-5426) or Neisseria meningitidis were adherent to cultured humanbuccal epithelial cell line (16HBE140) instead of HUVECs; fixed,blocked, incubated with MAb A4 and anti-capsular serogroup B antibodythen detected using fluorescently labeled secondary antibodies (TRITC orFITC).

(5) Fine structural analysis of purified O-deacylated LPS samples bynegative-ion ES-MS and NMR (Plested et al., 1999 IAI 67: 5417-5426).

Results:

1) Accessibility of Lps Epitope in Neisseria meningitidis Whole-Cells.

See FIGS. 7A-7E.

MAb A4 accesses the inner core LPS epitope in Neisseria meningitidis L4galE mutant in the presence of capsule (magnification ×100). Neisseriameningitidis L4 galE adherent to epithelial cells (16HBE140) stainedwith:

FIG. 7A: MAb A4 (anti-mouse TRITC-red);FIG. 7B: anti-cap B (anti-rabbit FITC-green);FIG. 7C: triple staining with MAb A4 (anti-mouse TRITC-red), anti-cap B(anti-rabbit FITC-green) and epithelial cells stained DAPI (blue).

MAb B5 accesses inner core LPS epitopes in Neisseria meningitidis L3MC58 (magnification ×2400). Neisseria meningitidis L3 MC58 adherent toHUVECs stained with:

FIG. 7D: MAb B5 (antimouse TRITC-red);FIG. 7E: anti-cap B (anti-rabbit FITC-green) using confocalimmunofluorescence microscopy.2) Conservation of Lps Epitope Across all Serogroup of Neisseriameningitidis

See FIG. 8

MAb A4 (diagonal hatched) and MAb B5 (horizontal lines) togetherrecognize all Neisseria meningitidis strains by immunoblotting withwhole-cell lysates, except 3 strains (black arrows) which are underfurther analysis. The dendrogram of genetic relationship of Neisseriameningitidis strains from a global collection was constructed by clusteranalysis following Multi-Locus Sequence Typing (MLST) (Maiden et al.,1998. PNAS 95: 3140-3145).

3) Genetically Defined LPS Structure See FIGS. 3A-3C.

Fine LPS structural details demonstrate conformational effects of PEtnon epitope presented. Space-filling 3-D molecular models of (MonopoliesMonte Carlo) calculated lowest energy states of core LPS from galEmutants FIG. 3A: L3; FIG. 3B: L4; FIG. 3C: L8 (dephosphorylated). Kdo ingrey, Heptose (Hep) in red, Glucose (Glc) and Glucosamine (GlcNAc) inlight and darker green, (PEtn) in brown.

Conclusions:

Inner core glycoforms have been identified with PEtn in the 3-positionof HepIi, an exocyclic position of Hep II or absent. This study hasindicated that utilization of MAb A4 in conjunction with MAb B5 enables97% of meningococcal strains to be recognized. These studies thereforeindicate that inner core LPS may have potential as a Neisseriameningitidis serogroup B vaccine.

Example 3 Studies on the Functional Activity of Monoclonal Antibody, MAbB5, and Inner Core (galE) Lipopolysaccharide Antibodies in Human SerumUsing an Opsonophagocytosis Assay, a Serum Bactericidal Assay and an InVivo Passive Protection Model Introduction

We have generated a monoclonal antibody, MAb B5. This antibody isaccessible to inner core LPS structures in Neisseria meningitidis in thepresence of capsule and is conserved in 70% of a representativecollection of Neisseria meningitidis of all strains and 76% of serogroupB strains (Plested, J. S. et al., 1999. Infect. Immun. 67 (10):5417-5426).

Until now it was not known if antibodies in a natural human infectioncan be specific for MAb B5 epitope and have functional activity.

MAb B5 has been shown to have opsonic and bactericidal activity againstgalE mutant and ability to passively protect infant rats againstchallenge with Neisseria meningitidis galE mutant using an in vivomodel.

Methods

(1) Opsonophagocytosis (OP) assay (Plested et al., 2000b): Briefly,fluorescently labeled ethanol-fixed Neisseria meningitidis MC58 or galEmutant or beads coated with purified galE LPS (10 μg/ml) were opsonisedwith MAb B5 and human complement source diluted in final buffer for 10mins/37° C./500 rpm in microtitre plate. Then human peripheral bloodpolymorphonuclear cells (PMNs) prepared from heparinized donor bloodwere diluted in final buffer and added to each well (1×10⁷ cells/ml) andincubated for a further 10 min/37° C./500 rpm. Reaction mixture wasstopped on ice by addition of 150 μl PBS-EDTA and added to FACS tubecontaining 50 μl TRYPAN BLUE. Mixture was mixed and 10,000 lymphocyteswere analysed on FACSCAN and CELLQUEST software. PMNs were analyzed byFSC vs appropriate channels to determine % uptake of fluorescentbacteria by granulocytes and monocytes (% OP activity).

(2) Serum Bactericidal (SB) assay method was adapted from CDC protocolexcept MAb B5 was added to dilutions of human pooled sera and 1000 cfuof Neisseria meningitidis strain and incubation time was 40-45 min at37° C. Briefly, bacteria were grown up onto BHI agar overnight fromfrozen stocks. A suspension of bacteria in PBS-B was measured atOD₂₆₀(1:50 in 1% SDS, 0.1% NaOH). Using a 96-well microtitre plate 50 μlbuffer was added to wells in columns 2-7. 50 μl of 80% decomplementedhuman pooled sera was added to column 8 wells. 100 μl of 80% pooled serawas added to wells in column 1. Two-fold serial dilutions of antibodywere added to columns 1-7 (discarding the last 500 from column 7). 500of bacterial suspension diluted to give 1000 cfu in 500 were added towells of columns 1-8. The mixture was incubated for 40-45 minutes andplated out onto BHI agar for overnight incubation. The number ofcolonies on each plate was counted and the results expressed as a % ofcfu/ml in decomplemented control well.

(3) In vivo passive protection model using 5-day old Wistar infant ratmodel. This model was as described by Moe, G. R., et al., 1999. Infect.Immun. 67: 5664-5675, except higher doses of Neisseria meningitidisbacteria were used and different Neisseria meningitidis strain was used.Briefly, groups of 5 day old infant rats were randomized with mothers.Weighed and given inoculum 1×10⁸ cfu/ml Neisseria meningitidis galEmutant mixed 1:1 with either (i) No antibody (PBS) (ii) Affinitypurified MAb B5 (10 μg) (iii) Affinity purified MAb B5(100 μg) (iv) MAb735 (anticapsular group B antibody) (49). Infant rats were monitored forsigns of infection and sampled by tail vein bleed at 6 hourspost-infection. Animals were weighed and terminal bleed was taken after24 h by cardiac puncture following injection of pentobarbitone. Neat anddiluted blood were plated immediately onto BHI plates and incubatedovernight. Plates were counted next day to determine bacteremia (cfu/ml)at 6 h and 24 h.

(4) LPS ELISA (Plested et. al., 2000a. Microtitre plates (NUNC) coatedwith purified (galE) LPS (10 μg/ml) overnight, were washed, blocked andincubated with MAb or human sera for 1 h, washed and detected withanti-mouse or anti-human IgG alkaline phosphatase and p-NPP(OD_(A405nm)).

(5) Affinity purified MAb B5. Spent culture supernatant from MAb B5 waspurified on Protein A-SEPHAROSE column and eluted with Glycine pH 4.0,neutralized with Tris-HCl pH9.0. Fractions were tested for reactivity onLPS ELISA, pooled and concentrated using Amicon-filter. Purity wasdetermined by SDS-PAGE gel and protein concentration was determined byOD and protein assay.

6) FACS surface labeling of Neisseria meningitidis bacteria. The methodwas adapted from Moe, G. R., at al., 1999. Infect. Immun. 67: 5664-5675)except no sodium azide was included in the blocking buffer step (Plestedet al., 2000b). To prepare labeled bacteria Neisseria meningitidis(strain MC58, galE) organisms were grown overnight by standardconditions at 37° C. on BHI agar plates and gently suspended in PBS.OD_(A260nm) was adjusted to give the required concentration e.g. 5×10⁹org./ml. 1000 bacterial cells were added to each FACS tube (5×10⁸ org.)and an equal volume of diluted sera (1/100 MAb B5 in 1% BSA/PBS) wasadded. Tubes were incubated for 2 hours at 4° C. and cells centrifugedfor 5 minutes at 13,000 g. The supernatant was discarded and cells werewashed with 2000 of 1% BSA/PBS. 1000 of FITC-conjugated F(ab)₂ goatanti-mouse (Sigma F2772) was added, diluted 1:100 in 1% BSMBS, and tubeswere incubated for 1 hour at 40 C. Cells were centrifuged at 13,000 gfor 5 minutes and washed by addition of 2000 of 1% BSMBS. Thesupernatant was discarded and the cells were suspended in 1% v/vformaldehyde. Samples were transferred to FACSCAN tubes and analyzed onthe FACS.

Results 1) Clinical Relevance of MAb 85 Epitope:

We present data on three paired sera taken from infants early (acute)and later (convalescent) during culture confirmed invasive meningococcaldisease (IMD) that resulted from infection with Neisseria meningitidisisolates of immunotypes L1, L3 (MAb B5 reactive) (patients 1 and 2) andL2 immunotype (MAb B5 non-reactive) (patient 3) (FIGS. 10A and 10B). TheNeisseria meningitidis isolates for patients 1, 2, 3 were L1 (B ntp1.14), L3 (B15 p1.7) and L2 (C2a p1.5) respectively. One paired serafrom patient 2 infected with a Neisseria meningitidis strain that wasMAb B5 reactive demonstrated an increase in specific inner core LPSantibodies by ELISA between early and late infection (p=0.03 notsignificant two-tailed paired t-test, 95% Cl 0.09-90.8)) (FIG. 10A).Patient 1 sera demonstrated no significant difference in the titre ofantibody taken early and later during IMD but the titer of the earlysample was already at a high level (FIG. 10A). The lack of increase mayreflect higher affinity antibody in the convalescent sample that wouldnot be detected in this ELISA. However in both patient 1 and 2 serathere was a nearly significant increase in functional activity in theconvalescent sera in an opsonophagocytosis assay with L3 wild-typestrain MC58 and human peripheral polymorphonuclear cells (p=0.06two-tailed paired t-test, 95% Cl 0.90-5.96) (FIG. 10B) (Plested et al.,2000b). There was no significant increase in specific antibody titrebetween acute and convalescent sera taken from patient 3 infected withL2 immunotype strain (MAb B5 non-reactive) as measured by ELISA (FIG.10A). There was no significant functional activity in OP assay againstL3 wild-type strain with sera taken from patient 3 early or later duringIMD (FIG. 10B). This demonstrates the clinical relevance of the MAb B5epitope in vivo and that specific inner core LPS antibodies arefunctional in vivo.

2) Supporting Evidence that Murine MAb 85 has Functional Activity inBiologically Relevant Assays and an In Vivo Model

(i) Opsonophagocytosis Assay

The OP assay provides evidence that MAb B5 has opsonic activity againstNeisseria meningitidis wild type and galE mutant and that the OPactivity is specific far MAb B5 epitope.

The specificity of MAb B5 reactivity using wild-type Neisseriameningitidis MC58 was shown by inhibition studies. MAb B5 waspre-incubated with different concentrations of purified LPS. There was adose response inhibition in OP activity with Neisseria meningitidis MC58with increasing concentrations of galE LPS added to MAb B5 (see FIG.11A).

MAb B5 has specific OP activity for MAb B5 reactive strains using anisogenic pair of Neisseria meningitidis wild-type strains (Neisseriameningitidis BZ157, serogroup B) that are MAb B5 reactive or MAb B5non-reactive. MAb B5 has opsonic activity with MAb B5 reactive strainbut not MAb B5 non-reactive strain (see FIG. 11B).

OP assay demonstrated the uptake of beads coated with purified L3 galELPS opsonised with MAb B5 was significantly greater than the uptake withuncoated beads. This demonstrates the specificity of MAb B5 for galE LPScoated onto beads (see FIG. 11C).

(ii) Serum Bactericidal Assay

The SB assay provides evidence that MAb B5 has bactericidal activityagainst Neisseria meningitidis galE mutant in SB assay in the presenceof a human complement source (see method).

The serum sensitivity of galE mutant with either no antibody or in thepresence of MAb B5 was compared (FIG. 12). There was a dose responseincrease in bactericidal activity of galE mutant shown by decreasing %survival, with decreasing % of serum in the presence of MAb B5 comparedto no antibody.

(iii) Passive Protection Model Using the Infant Rat.

Using the 5-day-old infant rat model we have demonstrated that two dosesMAb B5 are able to reduce bacteremia against challenge with 1×10⁸ cfu/mlNeisseria meningitidis MC58 galE mutant i.p. compared to no antibodycontrols. This data demonstrates the ability of MAb B5 to passivelyprotect against challenge with Neisseria meningitidis MC58 galE mutantand correlates with the functional activity of MAb B5 in OP and SBassays against the same Neisseria Meningitidis Strain, as Shown in FIG.13.

MAb 85 Binding Studies

Additional evidence that MAb B5 recognizes both wild-type and galEmutant LPS is shown in the following binding studies:

a) Western blot analysis

Purified LPS from wild type Neisseria meningitidis MC58 and galE mutantwas separated on standard Tricine gel and blotted onto nitrocellulose bystandard methods. The blot was probed with MAb B5 culture ascites(1:2000) overnight and detected using anti-mouse IgG and BCIP/NBTsubstrate. The blot demonstrates binding of MAb B5 to higher molecularweight wild-type LPS band and lower molecular weight galE LPS band inwild-type LPS. This demonstrates that MAb B5 can access and b i d to thewild-type LPS as well as truncated galE LPS, as shown in FIG. 14.

b) FACs Surface Labeling Data

MAb B5 binding to live wild-type strain MC58 and galE mutant, as shownin FIGS. 15A and 15B, respectively (1×10⁸ cfu/ml) were quantitativelycompared using surface labeling with anti-mouse FITC and analyzed byFACS. The relative binding of MAb B5 to Neisseria meningitidis MC58 was82.5% and Neisseria meningitidis galE mutant was 96.9% demonstratingthat as expected the greatest binding was to the galE mutant but therewas still significant binding to the wild-type strain MC58.

1. A vaccine for the treatment of disease caused by pathogenicNeisseria, the vaccine. comprising an immunogenic component based on theinner core of a Neisseria. lipopolysacchatide, LPS, and being capable ofeliciting functional antibodies against a majority of the strains withinthe species of the pathogenic Neisseria.
 2. A vaccine according to claim1, wherein the said immunogenic component is capable of elicitingfunctional antibodies against at least 60% of the strains within thespecies of the pathogenic Neisseria.
 3. A vaccine according to claim 2,wherein the said immunogenic component is capable of elicitingfunctional antibodies against at least 70% of the strains within thespecies of. the pathogenic Neisseria
 4. A vaccine according to anypreceding claim, wherein the immunogenic component is substantially freefrom outer core lipopolysaccharide
 5. A vaccine according to anypreceding claim, wherein the species of the pathogenic. Neisseria isNeisseria meningitidis.
 6. A vaccine according to claim 5, wherein theantibodies are elicited by the immunogenic component in at least 50% ofgroup B strains of Neisseria meningitidis.
 7. A vaccine according toclaim 5, wherein the antibodies are elicited by the immunogeniccomponent in at least 60% of group B strains of Neisseria meningitidis.8. A vaccine according to claim 5, wherein the antibodies are elicitedby the immunogenic component in at least 70% of group B strains ofNeisseria meningitidis.
 9. A vaccine according to any preceding claim,wherein the immunogenic component comprises of or consists of an epitopewhich is a part or all of the inner core structure of. a Neisseria LPS,is derived from this inner core, is a synthetic version of the innercore, or is a functional equivalent thereof.
 10. A vaccine according toany preceding claim, wherein the immunogenic component is an. epitope onthe LPS inner core characterised by the presence of aphosphoethanolamine. moiety linked to the 3-position at HepII of theinner core, or is a functional equivalent. thereof.
 11. A vaccineaccording to any preceding claim, wherein the immunogenic component isan. epitope on the LPS inner core which comprises a glucose residue atHepI.
 12. A vaccine according to any preceding claim, wherein theimmunogenic component is an. epitope on the LPS inner core whichcomprises an N-acetyl glucosamine at HepU of the. inner core LPS.
 13. Avaccine according to any preceding claim, wherein the inner core LPSconsists of an inner core oligosaccharide attached to lipid A, with thegeneral formula as show: Glc-P-(1,4)-Hyl-a-(1,s-) K do-a-(2,6)-Lipid A.where R1 is a substituent at the 3-position of HepII, and is hydrogen orGlc-α-(l, or. phosphoethanolamine; R2 is a substituent at the 6-positionof HepII, and is hydrogen or. phosphoethanolamine; R3 is a substituentat the 7-position of HepII, and is hydrogen or. phosphoethanolamine, andR4 is acetyl or hydrogen at the 3-position, 4-position or 6-position ofthe GlcNAc residue, or any combination thereof; and where Glc isDglucopymnose; Kdo is 3-deoxy-D-manno-2-octulosonic acid; Hep isL-glycero-D-manno-heptose, and GlcNAc is2-acetamido-2-deoxy-D-glucopyranose.
 14. A vaccine according to anypreceding claim, wherein the immunogenic component is. reactive with theB5 antibody produced by the hybridoma deposited under accession numberIDAC 260900-1.
 15. A vaccine comprising a few immunogenic componentsbased on the inner core of a. Neisseria lipopolysaccharide, LPS, andbeing capable of eliciting functional antibodies against a majority ofthe strains within the species of the pathogenic Neisseria.
 16. Avaccine according to claim 15 and including an immunogenic component asdefined in any of claims 1 to
 14. 17. A vaccine according to claim 15 or16, wherein the said few immunogenic components elicit functionalantibodies in at least 85% of the strains within the species of the.pathogenic Neisseria.
 18. A vaccine according to claim 17, wherein thesaid few immunogenic components elicit functional antibodies in at least95% of the strains within the species of the pathogenic. Neisseria. 19.A vaccine according to any of claims 15 to 18, wherein an immunogeniccomponent is reactive with the A4 antibody produced by the hybridomadeposited under accession. number IDAC 260900-2.
 20. A vaccine accordingto any preceding claim, wherein the immunogenic element of the vaccineis an epitope accessible on the bacterium in the presence of bacterialcapsule.
 21. A vaccine according to any preceding claim, comprising oneor more immunogen components which are capable of stimulating antibodieswhich are opsonic.
 22. A vaccine according to any preceding claim forthe treatment of Neisseria meningitidis
 23. A vaccine according to claim22 for the treatment of Neisseria meningitidis group B.
 24. A vaccineaccording to any preceding claim for the prevention of meningitissepticaemia or pneumonia or other manifestation of systemic or localdisease occasioned. by Neisseria meningitidis.
 25. A vaccine accordingto any of claims 1 to 22 for the treatment of urethritis, salpingitis,cervicitis, proctitis, pharyngitis, pelvic inflammatory disease or othermanifestation of. systemic or local disease occasioned by Neisseriagonorrhoeae.
 26. A vaccine according to any preceding claim which is aconjugated vaccine.
 27. A vaccine according to any preceding claim,which is derived from a commensal Neisseria.
 28. A vaccine according toclaim 27, wherein the commensal Neisseria is Neisseria lactomica.
 29. Anantibody reactive with an immunogenic component as defined in anypreceding claim.
 30. An antibody according to claim 29, wherein theantibody is humanized or otherwise customized to enhance suitability foradministration to a human.
 31. An antibody according to claim 29,obtainable from the hybridoma producing antibody B5
 32. An antibodyaccording to claim 29, obtainable from the hybridoma producing antibodyA4.
 33. A hybridoma producing antibody B5.
 34. A hybridoma producingantibody A4.
 35. A pharmaceutical preparation comprising an antibodyaccording to any of claims 29 to 32 in combination with apharmaceutically acceptable carrier.
 36. A method for the treatment ofNeisseria infection, the method comprising administering to a subject inneed of such treatment an effective amount of a vaccine according to anyof claims 1 to
 28. 37. A method for the treatment of Neisseriainfection, the method comprising administering to a subject in need ofsuch treatment an effective amount of an antibody according to any ofclaims 28 to
 31. 38. A method for the identification of immunogenicepitopes of strains of a species of Neisseria, the method comprising thesteps of generating antibodies to the inner core of a Neisseriabacterium, by inoculation of a host organism with a galE mutant strainof Neisseria meningifidis, and testing such antibodies against a wildtype Neisseria meningitidis strain to identify those antibodies whichare reactive, and for which the epitopes are therefore accessible. 39.Use of one or more biosynthetic pathway genes in the production of aNeisseria strain for the assessment, treatment or prevention ofNeisseria infection.
 40. Use of an immunogenic component, or a fewimmunogenic components, based on the inner core of a Neisserialipopolysaccharide, LPS, and being capable of eliciting functionalantibodies against a majority of the strains within the species of thepathogenic Neisseria, in the preparation of a medicament for thetreatment of a disease caused by a pathogenic Neisseria infection. 41.Use of an antibody according to any of claims 29 to 32 in thepreparation of a medicament for the treatment of Neisseria infection.